From 1ae1c41e393c6b174d74753f3ab3b184c071c034 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Wed, 30 Jul 2025 13:02:41 +0200 Subject: [PATCH 001/138] Use Freyja version 2.0.0 --- config/README.md | 10 ++++++++-- config/config.yaml | 6 ++++++ workflow/envs/freyja.yaml | 2 +- workflow/rules/demix.smk | 28 +++++++++++++++++++--------- 4 files changed, 34 insertions(+), 12 deletions(-) diff --git a/config/README.md b/config/README.md index 23e0367..8a1d7bb 100644 --- a/config/README.md +++ b/config/README.md @@ -141,10 +141,16 @@ All of the following variables are pre-defined in [config.yaml](/config/config.y - `IVAR_QUALITY`: minimum base quality for `ivar variants` (option `-q`). - `IVAR_FREQ`: minimum frequency threshold for `ivar variants` (option `-t`). - `IVAR_DEPTH`: minimum read depth for `ivar variants` (option `-m`). -- `DEMIX`: demixing configuration: +- `DEMIX`: demixing configuration (uses [Freyja](https://github.com/andersen-lab/Freyja), see also [its docs](https://andersen-lab.github.io/Freyja/index.html)): + - `PATHOGEN`: pathogen of interest for `freyja demix` (option `--pathogen`). - `MIN_QUALITY`: minimum quality for `freyja variants` (option `--minq`). - - `COV_CUTOFF`: minimum depth for `freyja demix` (option `--covcut`). + - `COV_CUTOFF`: minimum depth to calculate coverage for `freyja demix` (option `--covcut`). - `MIN_ABUNDANCE`: minimum lineage estimated abundance for `freyja demix` (option `--eps`). + - `CONFIRMED_ONLY`: exclude unconfirmed lineages in `freyja demix` (option `--confirmedonly`). + - `DEPTH_CUTOFF`: minimum depth on each site for `freyja demix` (option `--depthcutoff`). + - `RELAXED_MRCA`: assign clusters using relaxed (as opposed to strict) MRCA, used with `DEPTH_CUTOFF`, for `freyja demix` (option `--relaxedmrca`). + - `RELAXED_MRCA_THRESH`: `RELAXED_MRCA` threshold for `freyja demix` (option `--relaxedthresh`). + - `AUTO_ADAPT`: use error profile to set adaptive lasso penalty parameter for `freyja demix` (option `--autoadapt`). - `WINDOW`: sliding window of nucleotide variants per site configuration: - `WIDTH`: number of sites within windows. - `STEP`: number of sites between windows. diff --git a/config/config.yaml b/config/config.yaml index 7bb2fbe..e31b899 100644 --- a/config/config.yaml +++ b/config/config.yaml @@ -19,9 +19,15 @@ VC: IVAR_FREQ: 0.05 IVAR_DEPTH: 30 DEMIX: + PATHOGEN: "SARS-CoV-2" MIN_QUALITY: 20 COV_CUTOFF: 30 MIN_ABUNDANCE: 0.0001 + CONFIRMED_ONLY: false + DEPTH_CUTOFF: 30 + RELAXED_MRCA: false + RELAXED_MRCA_THRESH: 0.9 + AUTO_ADAPT: false WINDOW: WIDTH: 1000 STEP: 50 diff --git a/workflow/envs/freyja.yaml b/workflow/envs/freyja.yaml index 94d9be6..ff61e67 100644 --- a/workflow/envs/freyja.yaml +++ b/workflow/envs/freyja.yaml @@ -2,4 +2,4 @@ channels: - conda-forge - bioconda dependencies: - - freyja==1.4.2 + - freyja==2.0.0 diff --git a/workflow/rules/demix.smk b/workflow/rules/demix.smk index db6f6c2..654e2df 100644 --- a/workflow/rules/demix.smk +++ b/workflow/rules/demix.smk @@ -32,20 +32,30 @@ rule demix: variants_file = OUTDIR/"demixing"/"{sample}/{sample}_variants.tsv" params: coverage_cutoff = config["DEMIX"]["COV_CUTOFF"], - minimum_abundance = config["DEMIX"]["MIN_ABUNDANCE"] + minimum_abundance = config["DEMIX"]["MIN_ABUNDANCE"], + confirmed_only = "--confirmedonly " if config["DEMIX"]["CONFIRMED_ONLY"] else "", + depth_cutoff = config["DEMIX"]["DEPTH_CUTOFF"], + auto_adapt = "--autoadapt " if config["DEMIX"]["AUTO_ADAPT"] else "", + relaxed_mrca = "--relaxedmrca " if config["DEMIX"]["RELAXED_MRCA"] else "", + relaxed_mrca_thresh = config["DEMIX"]["RELAXED_MRCA_THRESH"], + pathogen = config["DEMIX"]["PATHOGEN"] output: demix_file = OUTDIR/"demixing"/"{sample}/{sample}_demixed.tsv" log: LOGDIR / "demix" / "{sample}.log.txt" shell: - """ - freyja demix \ - "{input.variants_file}" \ - "{input.depth_file}" \ - --eps {params.minimum_abundance} \ - --covcut {params.coverage_cutoff} \ - --output {output.demix_file} >{log} 2>&1 - """ + "freyja demix " + "{input.variants_file:q} " + "{input.depth_file:q} " + "--eps {params.minimum_abundance} " + "--covcut {params.coverage_cutoff} " + "--depthcutoff {params.depth_cutoff} " + "{params.confirmed_only}" + "{params.auto_adapt}" + "{params.relaxed_mrca}" + "--relaxedthresh {params.relaxed_mrca_thresh} " + "--output {output.demix_file} " + ">{log} 2>&1" rule summarise_demixing: From 7c52cae3fe7b0a41f13128fd1a5f3dee72c3e7b4 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Wed, 30 Jul 2025 14:25:38 +0200 Subject: [PATCH 002/138] Add Freyja data update --- config/README.md | 2 +- workflow/rules/demix.smk | 33 ++++++++++++++++++++++++++++++--- 2 files changed, 31 insertions(+), 4 deletions(-) diff --git a/config/README.md b/config/README.md index 8a1d7bb..667f276 100644 --- a/config/README.md +++ b/config/README.md @@ -142,7 +142,7 @@ All of the following variables are pre-defined in [config.yaml](/config/config.y - `IVAR_FREQ`: minimum frequency threshold for `ivar variants` (option `-t`). - `IVAR_DEPTH`: minimum read depth for `ivar variants` (option `-m`). - `DEMIX`: demixing configuration (uses [Freyja](https://github.com/andersen-lab/Freyja), see also [its docs](https://andersen-lab.github.io/Freyja/index.html)): - - `PATHOGEN`: pathogen of interest for `freyja demix` (option `--pathogen`). + - `PATHOGEN`: pathogen of interest for `freyja update` (option `--pathogen`); must be 'SARS-CoV-2'. - `MIN_QUALITY`: minimum quality for `freyja variants` (option `--minq`). - `COV_CUTOFF`: minimum depth to calculate coverage for `freyja demix` (option `--covcut`). - `MIN_ABUNDANCE`: minimum lineage estimated abundance for `freyja demix` (option `--eps`). diff --git a/workflow/rules/demix.smk b/workflow/rules/demix.smk index 654e2df..b5e8416 100644 --- a/workflow/rules/demix.smk +++ b/workflow/rules/demix.smk @@ -1,3 +1,25 @@ +rule demix_update: + threads: 1 + shadow: "shallow" + conda: + "../envs/freyja.yaml" + params: + pathogen = config["DEMIX"]["PATHOGEN"] + output: + folder = directory(OUTDIR/"demixing"/"freyja_data"), + curated_lineages = OUTDIR/"demixing"/"freyja_data"/"curated_lineages.json", + last_barcode_update = OUTDIR/"demixing"/"freyja_data"/"last_barcode_update.txt", + lineage_mutations = OUTDIR/"demixing"/"freyja_data"/"lineage_mutations.json", + lineage_yml = OUTDIR/"demixing"/"freyja_data"/"lineages.yml", + pathogens = OUTDIR/"demixing"/"freyja_data"/"pathogen_config.yml", + usher_barcodes = OUTDIR/"demixing"/"freyja_data"/"usher_barcodes.feather" + log: + LOGDIR / "demix_update" / "log.txt" + shell: + "mkdir -p {output.folder:q} && " + "freyja update --outdir {output.folder:q} --pathogen {params.pathogen:q} >{log} 2>&1" + + rule demix_preprocessing: threads: 1 conda: "../envs/freyja.yaml" @@ -29,7 +51,10 @@ rule demix: shadow: "minimal" input: depth_file = OUTDIR/"demixing"/"{sample}/{sample}_depth.txt", - variants_file = OUTDIR/"demixing"/"{sample}/{sample}_variants.tsv" + variants_file = OUTDIR/"demixing"/"{sample}/{sample}_variants.tsv", + barcodes = OUTDIR/"demixing"/"freyja_data"/"usher_barcodes.feather", + curated_lineages = OUTDIR/"demixing"/"freyja_data"/"curated_lineages.json", + lineage_yml = OUTDIR/"demixing"/"freyja_data"/"lineages.yml" params: coverage_cutoff = config["DEMIX"]["COV_CUTOFF"], minimum_abundance = config["DEMIX"]["MIN_ABUNDANCE"], @@ -37,8 +62,7 @@ rule demix: depth_cutoff = config["DEMIX"]["DEPTH_CUTOFF"], auto_adapt = "--autoadapt " if config["DEMIX"]["AUTO_ADAPT"] else "", relaxed_mrca = "--relaxedmrca " if config["DEMIX"]["RELAXED_MRCA"] else "", - relaxed_mrca_thresh = config["DEMIX"]["RELAXED_MRCA_THRESH"], - pathogen = config["DEMIX"]["PATHOGEN"] + relaxed_mrca_thresh = config["DEMIX"]["RELAXED_MRCA_THRESH"] output: demix_file = OUTDIR/"demixing"/"{sample}/{sample}_demixed.tsv" log: @@ -47,6 +71,9 @@ rule demix: "freyja demix " "{input.variants_file:q} " "{input.depth_file:q} " + "--barcodes {input.barcodes:q} " + "--meta {input.curated_lineages:q} " + "--lineageyml {input.lineage_yml:q} " "--eps {params.minimum_abundance} " "--covcut {params.coverage_cutoff} " "--depthcutoff {params.depth_cutoff} " From bab08de033ffa6b265113af5c1dadeed5382207a Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Wed, 30 Jul 2025 15:38:30 +0200 Subject: [PATCH 003/138] Update default depth cutoff --- config/config.yaml | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/config/config.yaml b/config/config.yaml index e31b899..61a7b83 100644 --- a/config/config.yaml +++ b/config/config.yaml @@ -24,7 +24,7 @@ DEMIX: COV_CUTOFF: 30 MIN_ABUNDANCE: 0.0001 CONFIRMED_ONLY: false - DEPTH_CUTOFF: 30 + DEPTH_CUTOFF: 0 RELAXED_MRCA: false RELAXED_MRCA_THRESH: 0.9 AUTO_ADAPT: false From 245ec94c377edf7b08c0e885812f116b359a3622 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Thu, 31 Jul 2025 10:41:34 +0200 Subject: [PATCH 004/138] Limit Freyja demix solver threads Ensures usage of a specific solver (Clarabel) by default and sets environment variables for all its internal linear system solvers --- config/config.yaml | 1 + workflow/rules/demix.smk | 12 ++++++++++-- 2 files changed, 11 insertions(+), 2 deletions(-) diff --git a/config/config.yaml b/config/config.yaml index 61a7b83..d3ef082 100644 --- a/config/config.yaml +++ b/config/config.yaml @@ -28,6 +28,7 @@ DEMIX: RELAXED_MRCA: false RELAXED_MRCA_THRESH: 0.9 AUTO_ADAPT: false + SOLVER: "CLARABEL" WINDOW: WIDTH: 1000 STEP: 50 diff --git a/workflow/rules/demix.smk b/workflow/rules/demix.smk index b5e8416..b40671d 100644 --- a/workflow/rules/demix.smk +++ b/workflow/rules/demix.smk @@ -54,7 +54,7 @@ rule demix: variants_file = OUTDIR/"demixing"/"{sample}/{sample}_variants.tsv", barcodes = OUTDIR/"demixing"/"freyja_data"/"usher_barcodes.feather", curated_lineages = OUTDIR/"demixing"/"freyja_data"/"curated_lineages.json", - lineage_yml = OUTDIR/"demixing"/"freyja_data"/"lineages.yml" + lineage_yml = OUTDIR/"demixing"/"freyja_data"/"lineages.yml", params: coverage_cutoff = config["DEMIX"]["COV_CUTOFF"], minimum_abundance = config["DEMIX"]["MIN_ABUNDANCE"], @@ -62,12 +62,19 @@ rule demix: depth_cutoff = config["DEMIX"]["DEPTH_CUTOFF"], auto_adapt = "--autoadapt " if config["DEMIX"]["AUTO_ADAPT"] else "", relaxed_mrca = "--relaxedmrca " if config["DEMIX"]["RELAXED_MRCA"] else "", - relaxed_mrca_thresh = config["DEMIX"]["RELAXED_MRCA_THRESH"] + relaxed_mrca_thresh = config["DEMIX"]["RELAXED_MRCA_THRESH"], + solver = config["DEMIX"]["SOLVER"], output: demix_file = OUTDIR/"demixing"/"{sample}/{sample}_demixed.tsv" log: LOGDIR / "demix" / "{sample}.log.txt" shell: + "RAYON_NUM_THREADS={threads} " + "JULIA_NUM_THREADS={threads} " + "BLAS_NUM_THREADS={threads} " + "OPENBLAS_NUM_THREADS={threads}" + "MKL_NUM_THREADS={threads} " + "OMP_NUM_THREADS={threads} " "freyja demix " "{input.variants_file:q} " "{input.depth_file:q} " @@ -81,6 +88,7 @@ rule demix: "{params.auto_adapt}" "{params.relaxed_mrca}" "--relaxedthresh {params.relaxed_mrca_thresh} " + "--solver {params.solver} " "--output {output.demix_file} " ">{log} 2>&1" From 0652c807052ad237af124c94b2b06039d4e73bf8 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Thu, 31 Jul 2025 13:50:05 +0200 Subject: [PATCH 005/138] Replace Freyja variants with equivalent samtools and ivar command --- workflow/rules/demix.smk | 22 ++++++++++------------ 1 file changed, 10 insertions(+), 12 deletions(-) diff --git a/workflow/rules/demix.smk b/workflow/rules/demix.smk index b40671d..06942c8 100644 --- a/workflow/rules/demix.smk +++ b/workflow/rules/demix.smk @@ -22,27 +22,25 @@ rule demix_update: rule demix_preprocessing: threads: 1 - conda: "../envs/freyja.yaml" + conda: "../envs/var_calling.yaml" shadow: "minimal" input: bam = get_input_bam, ref_fasta = lambda wildcards: select_mapping_references_fasta() params: - minq = config["DEMIX"]["MIN_QUALITY"] + minq = config["DEMIX"]["MIN_QUALITY"], output: depth_file = OUTDIR/"demixing"/"{sample}/{sample}_depth.txt", - variants_file = OUTDIR/"demixing"/"{sample}/{sample}_variants.tsv" + variants_file = OUTDIR/"demixing"/"{sample}/{sample}_variants.tsv", log: - LOGDIR / "demix_preprocessing" / "{sample}.log.txt" + pileup = LOGDIR / "demix_preprocessing" / "{sample}_pileup.log.txt", + ivar = LOGDIR / "demix_preprocessing" / "{sample}_ivar.log.txt", shell: - """ - freyja variants \ - "{input.bam}" \ - --variants {output.variants_file} \ - --depths {output.depth_file} \ - --minq {params.minq} \ - --ref {input.ref_fasta} >{log} 2>&1 - """ + "set -euo pipefail && " + "samtools mpileup -aa -A -d 600000 -Q {params.minq} -q 0 -B -f {input.ref_fasta:q} {input.bam:q} >sample.pileup 2>{log.pileup:q} && " + "ivar variants -p variants -q {params.minq} -r {input.ref_fasta:q} >{log.ivar:q} 2>&1 {output.depth_file:q} && " + "mv variants.tsv {output.variants_file:q}" rule demix: From e7106298c213625418870398d8a5de7a2fefdbbc Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Thu, 31 Jul 2025 16:19:49 +0200 Subject: [PATCH 006/138] Update option description --- config/README.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/config/README.md b/config/README.md index 667f276..2bd06d5 100644 --- a/config/README.md +++ b/config/README.md @@ -144,7 +144,7 @@ All of the following variables are pre-defined in [config.yaml](/config/config.y - `DEMIX`: demixing configuration (uses [Freyja](https://github.com/andersen-lab/Freyja), see also [its docs](https://andersen-lab.github.io/Freyja/index.html)): - `PATHOGEN`: pathogen of interest for `freyja update` (option `--pathogen`); must be 'SARS-CoV-2'. - `MIN_QUALITY`: minimum quality for `freyja variants` (option `--minq`). - - `COV_CUTOFF`: minimum depth to calculate coverage for `freyja demix` (option `--covcut`). + - `COV_CUTOFF`: minimum depth to calculate the reported "coverage" (percent of sites with that depth) for `freyja demix` (option `--covcut`). - `MIN_ABUNDANCE`: minimum lineage estimated abundance for `freyja demix` (option `--eps`). - `CONFIRMED_ONLY`: exclude unconfirmed lineages in `freyja demix` (option `--confirmedonly`). - `DEPTH_CUTOFF`: minimum depth on each site for `freyja demix` (option `--depthcutoff`). From a6c2fb3cc13d14a5ec9dfce0e8cae576c1d7f9ee Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Thu, 31 Jul 2025 16:42:16 +0200 Subject: [PATCH 007/138] Add Freyja barcode timestamp in report --- template.qmd | 10 ++++++++-- workflow/rules/report.smk | 8 +++++--- 2 files changed, 13 insertions(+), 5 deletions(-) diff --git a/template.qmd b/template.qmd index eb5b82b..49ced41 100644 --- a/template.qmd +++ b/template.qmd @@ -42,6 +42,7 @@ params: heat_tab: "" omega_plot: "" name: "" + freyja_ts: "" output-file: report.html --- @@ -100,6 +101,11 @@ if (params$use_bionj) { } else { dist.tree.algo <- "neighbor-joining (NJ)" } + +# Freyja timestamp +freyja.timestamp <- read_file(params$freyja_ts) %>% + as.POSIXct(., format = "%m_%d_%Y-%H-%M") %>% + strftime(., format = "%d %B %Y at %H:%M") ``` ## Summary of the target samples dataset @@ -141,10 +147,10 @@ table %>% ### Lineage admixture -The estimated lineage admixture for each sample has been calculated +The lineage admixture for each sample has been estimated using [Freyja](https://github.com/andersen-lab/Freyja). -![Estimated lineage admixture of each sample. +![Estimated lineage admixture of each sample (Freyja barcode timestamp: `r freyja.timestamp`). Samples in the X-axis are ordered chronologically, from more ancient to newer.](`r params$freyja`){#fig-freyja} ### Phylogenetic reconstruction diff --git a/workflow/rules/report.smk b/workflow/rules/report.smk index df45ea1..df8ced2 100644 --- a/workflow/rules/report.smk +++ b/workflow/rules/report.smk @@ -188,10 +188,11 @@ rule report: heat_table = report(REPORT_DIR_TABLES/"figure_10.csv"), evo = report(REPORT_DIR_PLOTS/"figure_11.png"), omega_plot = report(REPORT_DIR_PLOTS/"figure_12.png"), + freyja_ts = OUTDIR/"demixing"/"freyja_data"/"last_barcode_update.txt", value = OUTDIR/"diversity.json", stats_lm = OUTDIR/"stats.lm.json", table = OUTDIR/"summary_table.csv", - sum_nv = OUTDIR/"summary_nv.json" + sum_nv = OUTDIR/"summary_nv.json", params: workflow_version = get_repo_version(BASE_PATH.as_posix(), __version__), min_ivar_freq = config["VC"]["IVAR_FREQ"], @@ -199,7 +200,7 @@ rule report: shalrt_reps = config["SHALRT_REPS"], name = config["OUTPUT_NAME"], use_bionj = config["USE_BIONJ"], - cor_method = config["COR"]["METHOD"] + cor_method = config["COR"]["METHOD"], output: html = report(OUTDIR/f"{OUTPUT_NAME}.report.html") log: @@ -233,6 +234,7 @@ rule report: "sum_nv = '{input.sum_nv}', " "heat_tab = '{input.heat_table}', " "omega_plot = '{input.omega_plot}', " + "freyja_ts = '{input.freyja_ts}', " "name = '{params.name}'))\" " - ">{log} 2>&1 && " + ">{log:q} 2>&1 && " 'mv "$(dirname {input.qmd:q})/report.html" {output.html:q}' From 578fb6f98543f2f6e5614bdd457e31b64a49f70d Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Thu, 31 Jul 2025 17:31:47 +0200 Subject: [PATCH 008/138] Decouple demix plot from data Fix plot design source Update sample sorting Update demix table column names Fix column names Update temp variable name Update structure --- workflow/rules/demix.smk | 16 ++--- workflow/rules/report.smk | 21 ++++-- workflow/scripts/report/freyja_plot.R | 68 +++---------------- workflow/scripts/report/freyja_plot_data.R | 56 +++++++++++++++ .../{summary_demixing.R => summarise_demix.R} | 14 ++-- 5 files changed, 99 insertions(+), 76 deletions(-) create mode 100644 workflow/scripts/report/freyja_plot_data.R rename workflow/scripts/{summary_demixing.R => summarise_demix.R} (78%) diff --git a/workflow/rules/demix.smk b/workflow/rules/demix.smk index 06942c8..c16cc3e 100644 --- a/workflow/rules/demix.smk +++ b/workflow/rules/demix.smk @@ -1,4 +1,4 @@ -rule demix_update: +rule demix_barcode_update: threads: 1 shadow: "shallow" conda: @@ -14,7 +14,7 @@ rule demix_update: pathogens = OUTDIR/"demixing"/"freyja_data"/"pathogen_config.yml", usher_barcodes = OUTDIR/"demixing"/"freyja_data"/"usher_barcodes.feather" log: - LOGDIR / "demix_update" / "log.txt" + LOGDIR / "demix_barcode_update" / "log.txt" shell: "mkdir -p {output.folder:q} && " "freyja update --outdir {output.folder:q} --pathogen {params.pathogen:q} >{log} 2>&1" @@ -63,7 +63,7 @@ rule demix: relaxed_mrca_thresh = config["DEMIX"]["RELAXED_MRCA_THRESH"], solver = config["DEMIX"]["SOLVER"], output: - demix_file = OUTDIR/"demixing"/"{sample}/{sample}_demixed.tsv" + demix_file = OUTDIR/"demixing"/"samples"/"{sample}/{sample}_demixed.tsv" log: LOGDIR / "demix" / "{sample}.log.txt" shell: @@ -91,15 +91,15 @@ rule demix: ">{log} 2>&1" -rule summarise_demixing: +rule summarise_demix: threads: 1 conda: "../envs/renv.yaml" shadow: "shallow" input: - tables = expand(OUTDIR/"demixing"/"{sample}/{sample}_demixed.tsv", sample=iter_samples()) + tables = expand(OUTDIR/"demixing"/"samples"/"{sample}/{sample}_demixed.tsv", sample=iter_samples()) output: - summary_df = report(OUTDIR/"summary_freyja_demixing.csv") + summary_df = report(OUTDIR/"demixing"/"summary.csv") log: - LOGDIR / "summarise_demixing" / "log.txt" + LOGDIR / "summarise_demix" / "log.txt" script: - "../scripts/summary_demixing.R" + "../scripts/summarise_demix.R" diff --git a/workflow/rules/report.smk b/workflow/rules/report.smk index df8ced2..7c1ab06 100644 --- a/workflow/rules/report.smk +++ b/workflow/rules/report.smk @@ -49,16 +49,27 @@ rule diversity: "../scripts/report/diversity_plot.R" +rule freyja_plot_data: + conda: "../envs/renv.yaml" + input: + summary_demixing = OUTDIR/"demixing"/"summary.csv", + metadata = config["METADATA"] + output: + data = report(REPORT_DIR_TABLES/"freyja.csv") + log: + LOGDIR / "freyja_plot_data" / "log.txt" + script: + "../scripts/report/freyja_plot_data.R" + + rule freyja_plot: conda: "../envs/renv.yaml" params: design = config["PLOTS"] input: - summary_demixing = OUTDIR/"summary_freyja_demixing.csv", - metadata = config["METADATA"] + data = REPORT_DIR_TABLES/"freyja.csv" output: - fig = report(REPORT_DIR_PLOTS/"figure_1.png"), - table = report(REPORT_DIR_TABLES/"figure_1.csv") + plot = report(REPORT_DIR_PLOTS/"freyja.png") log: LOGDIR / "freyja_plot" / "log.txt" script: @@ -175,7 +186,7 @@ rule report: shadow: "shallow" input: qmd = Path(config["REPORT_QMD"]).resolve(), - freyja = report(REPORT_DIR_PLOTS/"figure_1.png"), + freyja = report(REPORT_DIR_PLOTS/"freyja.png"), tree_ml = report(REPORT_DIR_PLOTS/"figure_2.png"), diversity = report(REPORT_DIR_PLOTS/"figure_3.png"), fig_cor = report(REPORT_DIR_PLOTS/"figure_4.png"), diff --git a/workflow/scripts/report/freyja_plot.R b/workflow/scripts/report/freyja_plot.R index e698091..911e5a4 100644 --- a/workflow/scripts/report/freyja_plot.R +++ b/workflow/scripts/report/freyja_plot.R @@ -13,43 +13,17 @@ log_threshold(INFO) source(snakemake@params[["design"]]) # Read inputs -demix <- read_csv(snakemake@input[["summary_demixing"]]) - -# DATA PROCESSING -log_info("Obtaining main lineages") -main_lineages <- demix %>% - group_by(sample) %>% - top_n(1, abundances) %>% - ungroup() %>% - pull(lineages) %>% - unique() - -# Obtain sample names ordered by CollectionDate -metadata <- read_csv(snakemake@input[["metadata"]]) -date_order <- metadata %>% - arrange(CollectionDate) %>% - filter(ID %in% demix$sample) %>% - pull(ID) %>% - unique() - -# PLOT -log_info("Plotting summary demixing") -demix_plot <- demix %>% - mutate( - lineages = case_when( - lineages %in% main_lineages ~ lineages - ) - ) %>% - group_by(lineages, sample) %>% +log_info("Plotting") +p <- read_csv(snakemake@input$data) %>% mutate( - abundances = sum(abundances) + sample = reorder(sample, CollectionDate), + lineage = ifelse(is_main, lineage, NA) ) %>% - unique() %>% ggplot() + aes( - x = factor(sample, date_order), - y = as.numeric(abundances), - fill = lineages + x = sample, + y = abundance, + fill = lineage ) + scale_fill_viridis_d( na.value = "gray50", @@ -68,34 +42,12 @@ demix_plot <- demix %>% fill = "Lineage" ) +log_info("Saving plot") ggsave( - filename = snakemake@output[["fig"]], - plot = demix_plot, + filename = snakemake@output[["plot"]], + plot = p, width = 159.2, height = 119.4, units = "mm", dpi = 250 ) - - -# PLOT TABLES -log_info("Saving plot table") -demix %>% - mutate( - lineages = case_when( - lineages %in% main_lineages ~ lineages, - TRUE ~ "Other" - ) - ) %>% - group_by(sample, lineages) %>% - summarise(abundances = sum(abundances)) %>% - ungroup() %>% - left_join( - select( - metadata, - ID, - CollectionDate - ), - by = c("sample" = "ID") - ) %>% - write.csv(snakemake@output[["table"]], row.names = FALSE) diff --git a/workflow/scripts/report/freyja_plot_data.R b/workflow/scripts/report/freyja_plot_data.R new file mode 100644 index 0000000..2aafcde --- /dev/null +++ b/workflow/scripts/report/freyja_plot_data.R @@ -0,0 +1,56 @@ +#!/usr/bin/env Rscript + +# Write stdout and stderr to log file +log <- file(snakemake@log[[1]], open = "wt") +sink(log, type = "message") +sink(log, type = "output") + +library(tidyverse) +library(logger) +log_threshold(INFO) + +# Read inputs +demix <- read_csv(snakemake@input[["summary_demixing"]]) + +# Data processing +log_info("Obtaining main lineages") +main_lineages <- demix %>% + group_by(sample) %>% + top_n(1, abundance) %>% + ungroup() %>% + pull(lineage) %>% + unique() + +# Obtain sample names ordered by CollectionDate +log_info("Sorting dates") +metadata <- read_csv(snakemake@input[["metadata"]]) +date_order <- metadata %>% + arrange(CollectionDate) %>% + filter(ID %in% demix$sample) %>% + pull(ID) %>% + unique() + +# Build plot data +log_info("Building plot data") +plot.data <- demix %>% + group_by(lineage, sample) %>% + summarize(abundance = sum(abundance)) %>% + ungroup() %>% + left_join( + select( + metadata, + ID, + CollectionDate + ), + by = c("sample" = "ID") + ) %>% + mutate( + is_main = lineage %in% main_lineages + ) + +# Save plot data +log_info("Saving plot data") +write_csv( + plot.data, + snakemake@output[["data"]] +) diff --git a/workflow/scripts/summary_demixing.R b/workflow/scripts/summarise_demix.R similarity index 78% rename from workflow/scripts/summary_demixing.R rename to workflow/scripts/summarise_demix.R index 3ae246f..895354f 100644 --- a/workflow/scripts/summary_demixing.R +++ b/workflow/scripts/summarise_demix.R @@ -11,8 +11,8 @@ log_threshold(INFO) # Empty dataframe to be filled with data demix <- data.frame( - "lineages" = NA, - "abundances" = NA, + "lineage" = NA, + "abundance" = NA, "sample" = NA ) %>% filter(!is.na(sample)) @@ -23,21 +23,25 @@ lapply( function(tsv_file) { read_tsv( tsv_file, - col_names = c("variable", "valor"), + col_names = c("variable", "value"), show_col_types = FALSE ) %>% filter( - row_number() %in% c(3, 4) + variable %in% c("lineages", "abundances") ) %>% pivot_wider( names_from = variable, - values_from = valor + values_from = value ) %>% separate_rows( lineages, abundances, sep = " " ) %>% + rename( + lineage = lineages, + abundance = abundances + ) %>% mutate( sample = str_extract( basename(tsv_file), From d1351d7c59a1b61121650a807aeb2305af176aaa Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Fri, 1 Aug 2025 11:30:10 +0200 Subject: [PATCH 009/138] Update demixing names --- template.qmd | 6 ++--- workflow/rules/report.smk | 22 +++++++++---------- .../report/{freyja_plot.R => demix_plot.R} | 0 .../{freyja_plot_data.R => demix_plot_data.R} | 0 4 files changed, 14 insertions(+), 14 deletions(-) rename workflow/scripts/report/{freyja_plot.R => demix_plot.R} (100%) rename workflow/scripts/report/{freyja_plot_data.R => demix_plot_data.R} (100%) diff --git a/template.qmd b/template.qmd index 49ced41..7d5b469 100644 --- a/template.qmd +++ b/template.qmd @@ -25,7 +25,7 @@ params: use_bionj: "" cor_method: "" div: "" - freyja: "" + demix: "" tree: "" tempest: "" SNV: "" @@ -148,10 +148,10 @@ table %>% ### Lineage admixture The lineage admixture for each sample has been estimated -using [Freyja](https://github.com/andersen-lab/Freyja). +using [Freyja](https://github.com/andersen-lab/Freyja) (@fig-demix). ![Estimated lineage admixture of each sample (Freyja barcode timestamp: `r freyja.timestamp`). -Samples in the X-axis are ordered chronologically, from more ancient to newer.](`r params$freyja`){#fig-freyja} +Samples in the X-axis are ordered chronologically, from more ancient to newer.](`r params$demix`){#fig-demix} ### Phylogenetic reconstruction diff --git a/workflow/rules/report.smk b/workflow/rules/report.smk index 7c1ab06..79eb82f 100644 --- a/workflow/rules/report.smk +++ b/workflow/rules/report.smk @@ -49,31 +49,31 @@ rule diversity: "../scripts/report/diversity_plot.R" -rule freyja_plot_data: +rule demix_plot_data: conda: "../envs/renv.yaml" input: summary_demixing = OUTDIR/"demixing"/"summary.csv", metadata = config["METADATA"] output: - data = report(REPORT_DIR_TABLES/"freyja.csv") + data = report(REPORT_DIR_TABLES/"demix.csv") log: - LOGDIR / "freyja_plot_data" / "log.txt" + LOGDIR / "demix_plot_data" / "log.txt" script: - "../scripts/report/freyja_plot_data.R" + "../scripts/report/demix_plot_data.R" -rule freyja_plot: +rule demix_plot: conda: "../envs/renv.yaml" params: design = config["PLOTS"] input: - data = REPORT_DIR_TABLES/"freyja.csv" + data = REPORT_DIR_TABLES/"demix.csv" output: - plot = report(REPORT_DIR_PLOTS/"freyja.png") + plot = report(REPORT_DIR_PLOTS/"demix.png") log: - LOGDIR / "freyja_plot" / "log.txt" + LOGDIR / "demix_plot" / "log.txt" script: - "../scripts/report/freyja_plot.R" + "../scripts/report/demix_plot.R" rule general_NV_description: @@ -186,7 +186,7 @@ rule report: shadow: "shallow" input: qmd = Path(config["REPORT_QMD"]).resolve(), - freyja = report(REPORT_DIR_PLOTS/"freyja.png"), + demix = report(REPORT_DIR_PLOTS/"demix.png"), tree_ml = report(REPORT_DIR_PLOTS/"figure_2.png"), diversity = report(REPORT_DIR_PLOTS/"figure_3.png"), fig_cor = report(REPORT_DIR_PLOTS/"figure_4.png"), @@ -229,7 +229,7 @@ rule report: "use_bionj='{params.use_bionj}', " "cor_method='{params.cor_method}', " "div='{input.diversity}', " - "freyja ='{input.freyja}', " + "demix ='{input.demix}', " "tree = '{input.tree}', " "tempest = '{input.temest}', " "SNV = '{input.SNV}', " diff --git a/workflow/scripts/report/freyja_plot.R b/workflow/scripts/report/demix_plot.R similarity index 100% rename from workflow/scripts/report/freyja_plot.R rename to workflow/scripts/report/demix_plot.R diff --git a/workflow/scripts/report/freyja_plot_data.R b/workflow/scripts/report/demix_plot_data.R similarity index 100% rename from workflow/scripts/report/freyja_plot_data.R rename to workflow/scripts/report/demix_plot_data.R From 79bf475efc2eeba99395f07896f5360e27805c07 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Fri, 1 Aug 2025 11:38:32 +0200 Subject: [PATCH 010/138] Rename heatmap data rule --- workflow/rules/report.smk | 60 +++++++++++++++++++-------------------- 1 file changed, 30 insertions(+), 30 deletions(-) diff --git a/workflow/rules/report.smk b/workflow/rules/report.smk index 79eb82f..8980329 100644 --- a/workflow/rules/report.smk +++ b/workflow/rules/report.smk @@ -1,10 +1,37 @@ -rule heatmap: +rule demix_plot_data: + conda: "../envs/renv.yaml" + input: + summary_demixing = OUTDIR/"demixing"/"summary.csv", + metadata = config["METADATA"] + output: + data = report(REPORT_DIR_TABLES/"demix.csv") + log: + LOGDIR / "demix_plot_data" / "log.txt" + script: + "../scripts/report/demix_plot_data.R" + + +rule demix_plot: + conda: "../envs/renv.yaml" + params: + design = config["PLOTS"] + input: + data = REPORT_DIR_TABLES/"demix.csv" + output: + plot = report(REPORT_DIR_PLOTS/"demix.png") + log: + LOGDIR / "demix_plot" / "log.txt" + script: + "../scripts/report/demix_plot.R" + + +rule heatmap_plot_data: conda: "../envs/renv.yaml" input: vcf = OUTDIR/f"{OUTPUT_NAME}.masked.filtered.tsv", metadata = config["METADATA"] output: - table = report(REPORT_DIR_TABLES/"figure_10.csv") + table = report(REPORT_DIR_TABLES/"heatmap.csv") log: LOGDIR / "heatmap" / "log.txt" script: @@ -49,33 +76,6 @@ rule diversity: "../scripts/report/diversity_plot.R" -rule demix_plot_data: - conda: "../envs/renv.yaml" - input: - summary_demixing = OUTDIR/"demixing"/"summary.csv", - metadata = config["METADATA"] - output: - data = report(REPORT_DIR_TABLES/"demix.csv") - log: - LOGDIR / "demix_plot_data" / "log.txt" - script: - "../scripts/report/demix_plot_data.R" - - -rule demix_plot: - conda: "../envs/renv.yaml" - params: - design = config["PLOTS"] - input: - data = REPORT_DIR_TABLES/"demix.csv" - output: - plot = report(REPORT_DIR_PLOTS/"demix.png") - log: - LOGDIR / "demix_plot" / "log.txt" - script: - "../scripts/report/demix_plot.R" - - rule general_NV_description: conda: "../envs/renv.yaml" params: @@ -196,7 +196,7 @@ rule report: panel = report(REPORT_DIR_PLOTS/"figure_7.png"), tree = report(REPORT_DIR_PLOTS/"figure_8.png"), temest = report(REPORT_DIR_PLOTS/"figure_9.png"), - heat_table = report(REPORT_DIR_TABLES/"figure_10.csv"), + heat_table = report(REPORT_DIR_TABLES/"heatmap.csv"), evo = report(REPORT_DIR_PLOTS/"figure_11.png"), omega_plot = report(REPORT_DIR_PLOTS/"figure_12.png"), freyja_ts = OUTDIR/"demixing"/"freyja_data"/"last_barcode_update.txt", From a5612e720669c379c87ccc89001a71b04251a990 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Fri, 1 Aug 2025 11:41:02 +0200 Subject: [PATCH 011/138] Set plot size from params --- workflow/rules/report.smk | 3 +++ workflow/scripts/report/demix_plot.R | 4 ++-- 2 files changed, 5 insertions(+), 2 deletions(-) diff --git a/workflow/rules/report.smk b/workflow/rules/report.smk index 8980329..c6754f3 100644 --- a/workflow/rules/report.smk +++ b/workflow/rules/report.smk @@ -17,6 +17,9 @@ rule demix_plot: design = config["PLOTS"] input: data = REPORT_DIR_TABLES/"demix.csv" + params: + plot_width_mm = 159.2, + plot_height_mm = 119.4, output: plot = report(REPORT_DIR_PLOTS/"demix.png") log: diff --git a/workflow/scripts/report/demix_plot.R b/workflow/scripts/report/demix_plot.R index 911e5a4..fc49544 100644 --- a/workflow/scripts/report/demix_plot.R +++ b/workflow/scripts/report/demix_plot.R @@ -46,8 +46,8 @@ log_info("Saving plot") ggsave( filename = snakemake@output[["plot"]], plot = p, - width = 159.2, - height = 119.4, + width = snakemake@params$plot_width_mm, + height = snakemake@params$plot_height_mm, units = "mm", dpi = 250 ) From afbe502026a15f7106bdf4d7c8e51664009a864d Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Fri, 1 Aug 2025 13:05:31 +0200 Subject: [PATCH 012/138] Decouple diversity plot from data --- workflow/rules/report.smk | 44 ++++++--- workflow/scripts/report/diversity_data.R | 107 +++++++++++++++++++++ workflow/scripts/report/diversity_plot.R | 115 ++--------------------- 3 files changed, 146 insertions(+), 120 deletions(-) create mode 100644 workflow/scripts/report/diversity_data.R diff --git a/workflow/rules/report.smk b/workflow/rules/report.smk index c6754f3..26b1c07 100644 --- a/workflow/rules/report.smk +++ b/workflow/rules/report.smk @@ -14,12 +14,11 @@ rule demix_plot_data: rule demix_plot: conda: "../envs/renv.yaml" params: - design = config["PLOTS"] - input: - data = REPORT_DIR_TABLES/"demix.csv" - params: + design = config["PLOTS"], plot_width_mm = 159.2, plot_height_mm = 119.4, + input: + data = REPORT_DIR_TABLES/"demix.csv" output: plot = report(REPORT_DIR_PLOTS/"demix.png") log: @@ -58,23 +57,38 @@ rule window: "../scripts/window.py" -rule diversity: +rule diversity_data: threads: 4 conda: "../envs/renv.yaml" params: - design = config["PLOTS"], bootstrap_reps = config["DIVERSITY_REPS"], - plot_width = 159.2, - plot_height = 119.4 + aln_reference = config["ALIGNMENT_REFERENCE"], input: study_fasta = OUTDIR/"nextalign"/f"{OUTPUT_NAME}.aligned.masked.fasta", - context_fasta = OUTDIR/"context"/"nextalign"/"context_sequences.aligned.masked.fasta" + context_fasta = OUTDIR/"context"/"nextalign"/"context_sequences.aligned.masked.fasta", + output: + divs = report(REPORT_DIR_TABLES/"diversity.txt"), + json = temp(REPORT_DIR_TABLES/"diversity.json"), + log: + LOGDIR / "diversity_data" / "log.txt" + script: + "../scripts/report/diversity_data.R" + + +rule diversity_plot: + threads: 1 + conda: "../envs/renv.yaml" + params: + design = config["PLOTS"], + plot_width_mm = 159.2, + plot_height_mm = 119.4, + input: + divs = report(REPORT_DIR_TABLES/"diversity.txt"), + json = REPORT_DIR_TABLES/"diversity.json", output: - fig = report(REPORT_DIR_PLOTS/"figure_3.png"), - json = temp(OUTDIR/"diversity.json"), - table = REPORT_DIR_TABLES/"figure_3.csv" + plot = report(REPORT_DIR_PLOTS/"diversity.png"), log: - LOGDIR / "diversity" / "log.txt" + LOGDIR / "diversity_plot" / "log.txt" script: "../scripts/report/diversity_plot.R" @@ -191,7 +205,7 @@ rule report: qmd = Path(config["REPORT_QMD"]).resolve(), demix = report(REPORT_DIR_PLOTS/"demix.png"), tree_ml = report(REPORT_DIR_PLOTS/"figure_2.png"), - diversity = report(REPORT_DIR_PLOTS/"figure_3.png"), + diversity = report(REPORT_DIR_PLOTS/"diversity.png"), fig_cor = report(REPORT_DIR_PLOTS/"figure_4.png"), SNV = report(REPORT_DIR_PLOTS/"figure_5a.png"), SNV_spike = report(REPORT_DIR_PLOTS/"figure_5b.png"), @@ -203,7 +217,7 @@ rule report: evo = report(REPORT_DIR_PLOTS/"figure_11.png"), omega_plot = report(REPORT_DIR_PLOTS/"figure_12.png"), freyja_ts = OUTDIR/"demixing"/"freyja_data"/"last_barcode_update.txt", - value = OUTDIR/"diversity.json", + value = REPORT_DIR_TABLES/"diversity.json", stats_lm = OUTDIR/"stats.lm.json", table = OUTDIR/"summary_table.csv", sum_nv = OUTDIR/"summary_nv.json", diff --git a/workflow/scripts/report/diversity_data.R b/workflow/scripts/report/diversity_data.R new file mode 100644 index 0000000..e70b21f --- /dev/null +++ b/workflow/scripts/report/diversity_data.R @@ -0,0 +1,107 @@ +#!/usr/bin/env Rscript + +# Write stdout and stderr to log file +log <- file(snakemake@log[[1]], open = "wt") +sink(log, type = "message") +sink(log, type = "output") + +library(ape) +library(pegas) +library(future.apply) +library(tidyverse) +library(jsonlite) +library(logger) +log_threshold(INFO) + +# Pi calculation +nucleotide.diversity <- function(dna_object, record.names, sample.size) { + sample <- sample(record.names, sample.size, replace = FALSE) + dna_subset <- dna_object[record.names %in% sample] + nuc.div(dna_subset) +} + +# Parallel bootstrapping +boot.nd.parallel <- function(aln, sample.size, reps) { + record.names <- names(aln) + future_sapply( + 1:reps, + function(x) { + nucleotide.diversity(aln, record.names, sample.size) + }, + future.seed = TRUE + ) +} + +# Read outgroup/context alignment +log_info("Reading context") +gene_ex <- read.dna( + snakemake@input[["context_fasta"]], + format = "fasta", + as.matrix = FALSE +) +gene_ex <- gene_ex[ + !startsWith(names(gene_ex), snakemake@params$aln_reference) +] + +# Read target (study) alignment +log_info("Reading target alignment") +study_aln <- read.dna( + snakemake@input[["study_fasta"]], + format = "fasta", + as.matrix = FALSE +) +study_aln <- study_aln[ + !startsWith(names(study_aln), snakemake@params$aln_reference) +] + +# Diversity value for our samples +log_info("Calculating diversity value for studied samples") +diversity <- nuc.div(study_aln) + +# Perform bootstrap +log_info("Performing calculation for nucleotide diversity in context samples") +plan(multisession, workers = snakemake@threads) +divs <- boot.nd.parallel( + gene_ex, + length(study_aln), + snakemake@params[["bootstrap_reps"]] +) +plan(sequential) + +# Test normality +log_info("Normality test for nucleotide diversity values") +st <- shapiro.test(divs) + +# Calculate p-value (assuming normal distribution) +log_info("Calculating p-value (assuming normal distribution)") +test <- t.test( + divs, + alternative = "greater", + mu = diversity, + conf.level = 0.95 +) +pvalue.norm <- test$p.value + +# Estimate p-value empirically +log_info("Estimating p-value empirically") +empirical.probs <- ecdf(divs) +pvalue.emp <- empirical.probs(diversity) + +# Data for JSON file +log_info("Building JSON data") +p.value <- ifelse(st$p.value >= 0.05, pvalue.norm, pvalue.emp) +list.div <- list( + "diversity" = format(diversity, scientific = TRUE), + "p.value" = ifelse(p.value >= 0.001, p.value, "< 0.001"), + "normal.pvalue" = ifelse(st$p.value >= 0.001, p.value, "< 0.001"), + "norm.text" = ifelse(st$p.value >= 0.05, "", "not"), + "type.test" = ifelse(st$p.value >= 0.05, "", "empirical"), + "boot.reps" = snakemake@params[["bootstrap_reps"]], + "sample.size" = length(study_aln) +) + +log_info("Writing diversity distribution") +write_lines(divs, snakemake@output[["divs"]]) + +log_info("Writing results JSON") +write_json(list.div, snakemake@output[["json"]]) diff --git a/workflow/scripts/report/diversity_plot.R b/workflow/scripts/report/diversity_plot.R index 3ad0131..e50f7a7 100644 --- a/workflow/scripts/report/diversity_plot.R +++ b/workflow/scripts/report/diversity_plot.R @@ -5,94 +5,22 @@ log <- file(snakemake@log[[1]], open = "wt") sink(log, type = "message") sink(log, type = "output") -library(ape) -library(pegas) -library(future.apply) library(tidyverse) library(jsonlite) library(logger) log_threshold(INFO) -# Pi calculation -nucleotide.diversity <- function(dna_object, record.names, sample.size) { - sample <- sample(record.names, sample.size, replace = FALSE) - dna_subset <- dna_object[record.names %in% sample] - nuc.div(dna_subset) -} - -# Parallel bootstrapping -boot.nd.parallel <- function(aln, sample.size = 12, reps = 100) { - record.names <- names(aln) - future_sapply( - 1:reps, - function(x) { - nucleotide.diversity(aln, record.names, sample.size) - }, - future.seed = TRUE - ) -} - # Import file with plots style source(snakemake@params[["design"]]) -# Outgroup/context alignment -gene_ex <- read.dna( - snakemake@input[["context_fasta"]], - format = "fasta", - as.matrix = FALSE -) -gene_ex <- gene_ex[ - !startsWith(names(gene_ex), snakemake@config[["ALIGNMENT_REFERENCE"]]) -] - -# Study alignment -study_aln <- read.dna( - snakemake@input[["study_fasta"]], - format = "fasta", - as.matrix = FALSE -) -study_aln <- study_aln[ - !startsWith(names(study_aln), snakemake@config[["ALIGNMENT_REFERENCE"]]) -] - -# Diversity value for our samples -log_info("Calculating diversity value for studied samples") -diversity <- nuc.div(study_aln) - - -# Perform bootstrap -log_info("Performing calculation for nucleotide diversity in context samples") -plan(multisession, workers = snakemake@threads) -divs <- boot.nd.parallel( - gene_ex, - length(study_aln), - snakemake@params[["bootstrap_reps"]] -) -plan(sequential) - -# Test normality -log_info("Normality test for nucleotide diversity values") -st <- shapiro.test(divs) - -# Calculate p-value (assuming normal distribution) - -log_info("Calculating p-value (assuming normal distribution)") - -test <- t.test( - divs, - alternative = "greater", - mu = diversity, - conf.level = 0.95 -) -pvalue.norm <- test$p.value - -# Estimate p-value empirically -log_info("Estimating p-value empirically") -empirical.probs <- ecdf(divs) -pvalue.emp <- empirical.probs(diversity) +# Read data +log_info("Reading diversity results") +divs <- read_lines(snakemake@input$divs) %>% + as.numeric() +json <- read_json(snakemake@input$json) # Plot and save -log_info("Plotting diversity plot") +log_info("Plotting") p <- data.frame(pi = divs) %>% ggplot() + geom_density( @@ -103,7 +31,7 @@ p <- data.frame(pi = divs) %>% color = "#eae2b7" ) + geom_vline( - aes(xintercept = diversity), + xintercept = json$diversity, color = "#d62828" ) + stat_function( @@ -117,33 +45,10 @@ p <- data.frame(pi = divs) %>% ) ggsave( - filename = snakemake@output[["fig"]], + filename = snakemake@output[["plot"]], plot = p, - width = snakemake@params[["plot_width"]], - height = snakemake@params[["plot_height"]], + width = snakemake@params[["plot_width_mm"]], + height = snakemake@params[["plot_height_mm"]], units = "mm", dpi = 250 ) - -# DATA JSON ##### -p.value <- ifelse(st$p.value >= 0.05, pvalue.norm, pvalue.emp) - -list.div <- list( - "diversity" = format(diversity, scientific = TRUE), - "p.value" = ifelse(p.value >= 0.001, p.value, "< 0.001"), - "normal.pvalue" = ifelse(st$p.value >= 0.001, p.value, "< 0.001"), - "norm.text" = ifelse(st$p.value >= 0.05, "", "not"), - "type.test" = ifelse(st$p.value >= 0.05, "", "empirical"), - "boot.reps" = snakemake@params[["bootstrap_reps"]], - "sample.size" = length(study_aln) -) - -json <- toJSON(list.div) -write(json, snakemake@output[["json"]]) - -# PLOT TABLES -data.frame( - pi = divs, - prop.value = diversity -) %>% - write.csv(snakemake@output[["table"]], row.names = FALSE) From 8ec0642e92a3134ff699ab049be04b95144c70c0 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Fri, 1 Aug 2025 13:36:39 +0200 Subject: [PATCH 013/138] Define diversity plot palette in design script --- config/design_plots.R | 14 ++++++++++---- workflow/scripts/report/diversity_plot.R | 8 ++++---- 2 files changed, 14 insertions(+), 8 deletions(-) diff --git a/config/design_plots.R b/config/design_plots.R index 971265c..fd10749 100644 --- a/config/design_plots.R +++ b/config/design_plots.R @@ -25,9 +25,8 @@ theme_update( panel.grid = element_line(linewidth = 0.17, color = "lightgray") ) - -# CONFIG -gene_colors = c( +# Gene palette +gene_colors <- c( M = "#B4D4B4", N = "#B7B7B8", orf1ab = "#9CC4DC", @@ -40,9 +39,16 @@ gene_colors = c( ORF10 = "#CACB5D" ) +# Nucleotide diversity +DIVERSITY_PALETTE <- c( + density_fill = "#fcbf49", + density_color = "#eae2b7", + value_color = "#d62828", + dnorm_color = "#f77f00" +) # M-L tree colors and labels -tree_colors = c( +tree_colors <- c( tip_label = "#D944AA99", boot_alrt_pass = "#64ACEEB2" ) diff --git a/workflow/scripts/report/diversity_plot.R b/workflow/scripts/report/diversity_plot.R index e50f7a7..b6c4c9c 100644 --- a/workflow/scripts/report/diversity_plot.R +++ b/workflow/scripts/report/diversity_plot.R @@ -25,19 +25,19 @@ p <- data.frame(pi = divs) %>% ggplot() + geom_density( aes(x = pi), - fill = "#fcbf49", + fill = DIVERSITY_PALETTE["density_fill"], alpha = 0.7, bw = 0.000001, - color = "#eae2b7" + color = DIVERSITY_PALETTE["density_color"] ) + geom_vline( xintercept = json$diversity, - color = "#d62828" + color = DIVERSITY_PALETTE["value_color"] ) + stat_function( fun = dnorm, args = list(mean = mean(divs), sd = sd(divs)), - color = "#f77f00" + color = DIVERSITY_PALETTE["dnorm_color"] ) + labs( x = "π", From 3af3f874bd7c7778f001b3a0277f79236c06c05d Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Fri, 1 Aug 2025 13:42:32 +0200 Subject: [PATCH 014/138] Update design variable names --- config/design_plots.R | 16 ++++++++-------- workflow/scripts/report/NV_description.R | 14 +++++++------- workflow/scripts/report/evo_plots.R | 8 ++++---- workflow/scripts/report/phylo_plots.R | 4 ++-- 4 files changed, 21 insertions(+), 21 deletions(-) diff --git a/config/design_plots.R b/config/design_plots.R index fd10749..91607e1 100644 --- a/config/design_plots.R +++ b/config/design_plots.R @@ -26,7 +26,7 @@ theme_update( ) # Gene palette -gene_colors <- c( +GENE_PALETTE <- c( M = "#B4D4B4", N = "#B7B7B8", orf1ab = "#9CC4DC", @@ -48,18 +48,18 @@ DIVERSITY_PALETTE <- c( ) # M-L tree colors and labels -tree_colors <- c( +TREE_PALETTE <- c( tip_label = "#D944AA99", boot_alrt_pass = "#64ACEEB2" ) -node.size <- c( +TREE_NODE_SIZE <- c( tip_label = 2, boot_alrt_pass = 0.8 ) # Nucleotide variants classification colors and labels -NV_colors <- c( +NV_TYPE_PALETTE <- c( Frameshift = "#568D63", "In frame" = "black", Intergenic = "#B27CF9", @@ -67,7 +67,7 @@ NV_colors <- c( Yes = "#0248FD" ) -NV_names <- c( +NV_TYPE_NAMES <- c( Frameshift = "Frameshift", "In frame" = "Inframe", Intergenic = "Intergenic", @@ -76,17 +76,17 @@ NV_names <- c( ) # dn ds colors and labels -dnds.labels <- c( +DNDS_LABELS <- c( dn = "dN", ds = "dS" ) -dnds.colors <- c( +DNDS_COLORS <- c( dn = "#E53E47", ds = "#2C47F5" ) -dnds.shapes <- c( +DNDS_SHAPES <- c( dn = 2, ds = 4 ) diff --git a/workflow/scripts/report/NV_description.R b/workflow/scripts/report/NV_description.R index d3e72fd..210e2f4 100644 --- a/workflow/scripts/report/NV_description.R +++ b/workflow/scripts/report/NV_description.R @@ -189,11 +189,11 @@ variants <- vcf %>% inherit.aes = FALSE, width = 0.3 ) + - scale_fill_manual(values = gene_colors) + + scale_fill_manual(values = GENE_PALETTE) + xlim(c(0, 29903)) + scale_color_manual( - labels = NV_names, - values = NV_colors + labels = NV_TYPE_NAMES, + values = NV_TYPE_PALETTE ) + scale_y_discrete(drop = FALSE) + labs( @@ -227,7 +227,7 @@ window_plot <- window %>% limits = c(0, max(window$fractions) + 0.005) ) + xlim(c(0, 29903)) + - scale_color_manual(values = gene_colors) + + scale_color_manual(values = GENE_PALETTE) + labs( y = "Proportion of \n sites with NV", x = "", @@ -312,7 +312,7 @@ window_plot_spike <- window %>% ) + xlim(c(min(spike_pos), max(spike_pos))) + scale_color_manual( - values = gene_colors, + values = GENE_PALETTE, guide = "none" ) + labs( @@ -337,8 +337,8 @@ variants_spike <- vcf_spike %>% geom_point(size = 3) + xlim(c(min(spike_pos), max(spike_pos))) + scale_color_manual( - labels = NV_names, - values = NV_colors + labels = NV_TYPE_NAMES, + values = NV_TYPE_PALETTE ) + scale_y_discrete(drop = FALSE) + labs( diff --git a/workflow/scripts/report/evo_plots.R b/workflow/scripts/report/evo_plots.R index e0ed984..31da73b 100644 --- a/workflow/scripts/report/evo_plots.R +++ b/workflow/scripts/report/evo_plots.R @@ -82,13 +82,13 @@ plot <- plot_df %>% geom_line() + scale_color_manual( name = "Parameter", - labels = dnds.labels, - values = dnds.colors + labels = DNDS_LABELS, + values = DNDS_COLORS ) + scale_shape_manual( name = "Parameter", - values = dnds.shapes, - labels = dnds.labels + values = DNDS_SHAPES, + labels = DNDS_LABELS ) + labs( y = "Substitution rate", diff --git a/workflow/scripts/report/phylo_plots.R b/workflow/scripts/report/phylo_plots.R index 447d873..dc1a622 100644 --- a/workflow/scripts/report/phylo_plots.R +++ b/workflow/scripts/report/phylo_plots.R @@ -242,11 +242,11 @@ p <- ggtree(tree_ml, layout = "circular") %<+% ml.tree.annot + geom_rootedge(0.05 * tree_ml.max.tip.length) + xlim(-tree_ml.max.tip.length / 3, NA) + scale_color_manual( - values = setNames(tree_colors[names(legend.names)], legend.names), + values = setNames(TREE_PALETTE[names(legend.names)], legend.names), na.translate = FALSE ) + scale_size_manual( - values = setNames(node.size[names(legend.names)], legend.names), + values = setNames(TREE_NODE_SIZE[names(legend.names)], legend.names), na.translate = FALSE ) From d66bc4ad49230ddcc93f074bea6c906371b120b2 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Fri, 1 Aug 2025 13:46:45 +0200 Subject: [PATCH 015/138] Update JSON writing --- workflow/scripts/report/diversity_data.R | 6 +++++- 1 file changed, 5 insertions(+), 1 deletion(-) diff --git a/workflow/scripts/report/diversity_data.R b/workflow/scripts/report/diversity_data.R index e70b21f..a6daa7d 100644 --- a/workflow/scripts/report/diversity_data.R +++ b/workflow/scripts/report/diversity_data.R @@ -104,4 +104,8 @@ log_info("Writing diversity distribution") write_lines(divs, snakemake@output[["divs"]]) log_info("Writing results JSON") -write_json(list.div, snakemake@output[["json"]]) +write_json( + list.div, + snakemake@output[["json"]], + auto_unbox = TRUE +) From dcd87a27af2fc017b820a03fcfa3f2329b447b2c Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Fri, 1 Aug 2025 13:54:18 +0200 Subject: [PATCH 016/138] Remove diversity formatting --- template.qmd | 2 +- workflow/scripts/report/diversity_data.R | 2 +- 2 files changed, 2 insertions(+), 2 deletions(-) diff --git a/template.qmd b/template.qmd index 7d5b469..abf0466 100644 --- a/template.qmd +++ b/template.qmd @@ -175,7 +175,7 @@ from the context dataset. The distribution of the nuclotide diversity is assumed `r div_values[["norm.text"]]` be normal after performing a Shapiro-Wilk test (p-value of $`r div_values[["normal.pvalue"]]`$). -The nucleotide diversity of the target samples is $`r div_values[["diversity"]] `$ (red line in @fig-div). +The nucleotide diversity of the target samples is $`r format(div_values[["diversity"]], scientific = TRUE) `$ (red line in @fig-div). Assuming the independence of the context samples, the `r div_values[["type.test"]]` p-value of the context samples having a nucleotide diversity (in orange in @fig-div) as low as that of the target dataset is $`r div_values[["p.value"]]`$. diff --git a/workflow/scripts/report/diversity_data.R b/workflow/scripts/report/diversity_data.R index a6daa7d..56fc5ab 100644 --- a/workflow/scripts/report/diversity_data.R +++ b/workflow/scripts/report/diversity_data.R @@ -91,7 +91,7 @@ pvalue.emp <- empirical.probs(diversity) log_info("Building JSON data") p.value <- ifelse(st$p.value >= 0.05, pvalue.norm, pvalue.emp) list.div <- list( - "diversity" = format(diversity, scientific = TRUE), + "diversity" = diversity, "p.value" = ifelse(p.value >= 0.001, p.value, "< 0.001"), "normal.pvalue" = ifelse(st$p.value >= 0.001, p.value, "< 0.001"), "norm.text" = ifelse(st$p.value >= 0.05, "", "not"), From f62211802c5355418da1b29a665e9e895bd164f5 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Fri, 1 Aug 2025 13:58:26 +0200 Subject: [PATCH 017/138] Remove temp directives --- workflow/rules/report.smk | 6 +++--- 1 file changed, 3 insertions(+), 3 deletions(-) diff --git a/workflow/rules/report.smk b/workflow/rules/report.smk index 26b1c07..7b81e2e 100644 --- a/workflow/rules/report.smk +++ b/workflow/rules/report.smk @@ -67,8 +67,8 @@ rule diversity_data: study_fasta = OUTDIR/"nextalign"/f"{OUTPUT_NAME}.aligned.masked.fasta", context_fasta = OUTDIR/"context"/"nextalign"/"context_sequences.aligned.masked.fasta", output: - divs = report(REPORT_DIR_TABLES/"diversity.txt"), - json = temp(REPORT_DIR_TABLES/"diversity.json"), + divs = REPORT_DIR_TABLES/"diversity.txt", + json = REPORT_DIR_TABLES/"diversity.json", log: LOGDIR / "diversity_data" / "log.txt" script: @@ -83,7 +83,7 @@ rule diversity_plot: plot_width_mm = 159.2, plot_height_mm = 119.4, input: - divs = report(REPORT_DIR_TABLES/"diversity.txt"), + divs = REPORT_DIR_TABLES/"diversity.txt", json = REPORT_DIR_TABLES/"diversity.json", output: plot = report(REPORT_DIR_PLOTS/"diversity.png"), From 5375a04e0f0c05487448a60211e03b40740f323c Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Fri, 1 Aug 2025 14:04:55 +0200 Subject: [PATCH 018/138] Avoid float rounding before writing JSON --- workflow/scripts/report/diversity_data.R | 3 ++- 1 file changed, 2 insertions(+), 1 deletion(-) diff --git a/workflow/scripts/report/diversity_data.R b/workflow/scripts/report/diversity_data.R index 56fc5ab..d852e8d 100644 --- a/workflow/scripts/report/diversity_data.R +++ b/workflow/scripts/report/diversity_data.R @@ -107,5 +107,6 @@ log_info("Writing results JSON") write_json( list.div, snakemake@output[["json"]], - auto_unbox = TRUE + auto_unbox = TRUE, + digits = NA # maximum precision ) From 3e18cd69ba46a24ceccdaecb1e2bbf6c056f7cae Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Fri, 1 Aug 2025 14:10:44 +0200 Subject: [PATCH 019/138] Avoid mentioning colors for flexibility --- template.qmd | 8 ++++---- 1 file changed, 4 insertions(+), 4 deletions(-) diff --git a/template.qmd b/template.qmd index abf0466..bcdd4e4 100644 --- a/template.qmd +++ b/template.qmd @@ -175,15 +175,15 @@ from the context dataset. The distribution of the nuclotide diversity is assumed `r div_values[["norm.text"]]` be normal after performing a Shapiro-Wilk test (p-value of $`r div_values[["normal.pvalue"]]`$). -The nucleotide diversity of the target samples is $`r format(div_values[["diversity"]], scientific = TRUE) `$ (red line in @fig-div). +The nucleotide diversity of the target samples is $`r format(div_values[["diversity"]], scientific = TRUE) `$ (vertical line in @fig-div). Assuming the independence of the context samples, the `r div_values[["type.test"]]` -p-value of the context samples having a nucleotide diversity (in orange in @fig-div) +p-value of the context samples having a nucleotide diversity (smooth curve in @fig-div) as low as that of the target dataset is $`r div_values[["p.value"]]`$. -![Analysis of the nucleotide diversity (π). The orange line describes +![Analysis of the nucleotide diversity (π). The smooth curve describes a normal distribution with the same mean and standard deviation as the distribution of π from $`r div_values[["boot.reps"]]`$ subsets of $`r div_values[["sample.size"]]`$ -sequences from the context. The red vertical line indicates the π value of the +sequences from the context. The vertical line indicates the π value of the target samples.](`r params$div`){#fig-div} ## Evolutionary trajectory of the serially-sampled SARS-CoV-2 infection From c4bc02aa3f901019376df9ef01387288fd37cefd Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Thu, 7 Aug 2025 12:21:08 +0200 Subject: [PATCH 020/138] Decouple context phylogeny plot from data --- config/design_plots.R | 11 ++ template.qmd | 8 +- workflow/rules/distances.smk | 2 +- workflow/rules/report.smk | 60 +++++++++- .../scripts/report/context_phylogeny_data.R | 104 ++++++++++++++++++ .../scripts/report/context_phylogeny_plot.R | 61 ++++++++++ workflow/scripts/report/phylo_plots.R | 98 +---------------- 7 files changed, 242 insertions(+), 102 deletions(-) create mode 100644 workflow/scripts/report/context_phylogeny_data.R create mode 100644 workflow/scripts/report/context_phylogeny_plot.R diff --git a/config/design_plots.R b/config/design_plots.R index 91607e1..166577f 100644 --- a/config/design_plots.R +++ b/config/design_plots.R @@ -58,6 +58,17 @@ TREE_NODE_SIZE <- c( boot_alrt_pass = 0.8 ) +TREE_LEGEND_NAMES <- c( + tip_label = "Target samples", + boot_alrt_pass = sprintf( + "UFBoot ≥ %s%s & SH-aLRT ≥ %s%s ", + snakemake@params[["boot_th"]], + "%", + snakemake@params[["alrt_th"]], + "%" + ) +) + # Nucleotide variants classification colors and labels NV_TYPE_PALETTE <- c( Frameshift = "#568D63", diff --git a/template.qmd b/template.qmd index bcdd4e4..8228e97 100644 --- a/template.qmd +++ b/template.qmd @@ -37,6 +37,7 @@ params: tree_ml: "" fig_cor_snp: "" stats_lm: "" + stats_ml: "" table: "" sum_nv: "" heat_tab: "" @@ -63,8 +64,11 @@ library(readr) # Diversity div_values <- fromJSON(params$div_value) -# Temporal signal -stats <- fromJSON(params$stats_lm) +# Temporal signal and context tree +stats <- append( + fromJSON(params$stats_lm), + fromJSON(params$stats_ml) +) correlation <- stats[["r2"]] sub_rate <- stats[["sub_rate"]] sub_rate <- round(sub_rate, digits = 2) diff --git a/workflow/rules/distances.smk b/workflow/rules/distances.smk index 4161df9..886d6a4 100644 --- a/workflow/rules/distances.smk +++ b/workflow/rules/distances.smk @@ -10,7 +10,7 @@ rule weighted_distances: ancestor = OUTDIR/f"{OUTPUT_NAME}.ancestor.fasta", reference = OUTDIR/"reference.fasta" output: - distances = REPORT_DIR_TABLES/f"figure_8.csv" + distances = REPORT_DIR_TABLES/f"distances.csv" log: LOGDIR / "weighted_distances" / "log.txt" script: diff --git a/workflow/rules/report.smk b/workflow/rules/report.smk index 7b81e2e..7e2c64d 100644 --- a/workflow/rules/report.smk +++ b/workflow/rules/report.smk @@ -120,6 +120,59 @@ rule general_NV_description: "../scripts/report/NV_description.R" +rule context_phylogeny_data: + conda: "../envs/renv.yaml" + params: + design = config["PLOTS"], + ref_name = config["ALIGNMENT_REFERENCE"], + boot_th = 95.0, + alrt_th = 80.0, + input: + target_fasta = OUTDIR/f"{OUTPUT_NAME}.fasta", + tree = OUTDIR/f"tree_context/{OUTPUT_NAME}.treefile", + output: + json = REPORT_DIR_TABLES/"context_phylogeny.json", + annotation = REPORT_DIR_TABLES/"context_phylogeny.csv", + log: + LOGDIR / "context_phylogeny_data" / "log.txt" + script: + "../scripts/report/context_phylogeny_data.R" + + +rule context_phylogeny_plot: + conda: "../envs/renv.yaml" + params: + design = config["PLOTS"], + plot_height_mm = 119.4, + plot_width_mm = 159.2, + input: + tree = OUTDIR/f"tree_context/{OUTPUT_NAME}.treefile", + json = REPORT_DIR_TABLES/"context_phylogeny.json", + annotation = REPORT_DIR_TABLES/"context_phylogeny.csv" + output: + plot = report(REPORT_DIR_PLOTS/"context_phylogeny.png"), + log: + LOGDIR / "context_phylogeny_plot" / "log.txt" + script: + "../scripts/report/context_phylogeny_plot.R" + + +# rule allele_freq_phylogeny_data: # nj +# ... + + +# rule allele_freq_phylogeny_plot: +# ... + + +# rule time_signal_data: # tempest +# ... + + +# rule time_signal_plot: # tempest +# ... + + rule phylo_plots: conda: "../envs/renv.yaml" params: @@ -131,14 +184,13 @@ rule phylo_plots: plot_width_mm = 159.2, use_bionj = config["USE_BIONJ"] input: - dist = REPORT_DIR_TABLES/f"figure_8.csv", + dist = REPORT_DIR_TABLES/f"distances.csv", study_fasta = OUTDIR/f"{OUTPUT_NAME}.fasta", ml = OUTDIR/f"tree_context/{OUTPUT_NAME}.treefile", metadata = config["METADATA"] output: temest = report(REPORT_DIR_PLOTS/"figure_9.png"), tree = report(REPORT_DIR_PLOTS/"figure_8.png"), - tree_ml = report(REPORT_DIR_PLOTS/"figure_2.png"), table = report(REPORT_DIR_TABLES/"figure_9.csv"), json = temp(OUTDIR/"stats.lm.json") log: @@ -204,7 +256,7 @@ rule report: input: qmd = Path(config["REPORT_QMD"]).resolve(), demix = report(REPORT_DIR_PLOTS/"demix.png"), - tree_ml = report(REPORT_DIR_PLOTS/"figure_2.png"), + tree_ml = report(REPORT_DIR_PLOTS/"context_phylogeny.png"), diversity = report(REPORT_DIR_PLOTS/"diversity.png"), fig_cor = report(REPORT_DIR_PLOTS/"figure_4.png"), SNV = report(REPORT_DIR_PLOTS/"figure_5a.png"), @@ -219,6 +271,7 @@ rule report: freyja_ts = OUTDIR/"demixing"/"freyja_data"/"last_barcode_update.txt", value = REPORT_DIR_TABLES/"diversity.json", stats_lm = OUTDIR/"stats.lm.json", + stats_ml = REPORT_DIR_TABLES/"context_phylogeny.json", table = OUTDIR/"summary_table.csv", sum_nv = OUTDIR/"summary_nv.json", params: @@ -258,6 +311,7 @@ rule report: "tree_ml = '{input.tree_ml}', " "fig_cor_snp = '{input.fig_cor}', " "stats_lm = '{input.stats_lm}', " + "stats_ml = '{input.stats_ml}', " "table = '{input.table}', " "sum_nv = '{input.sum_nv}', " "heat_tab = '{input.heat_table}', " diff --git a/workflow/scripts/report/context_phylogeny_data.R b/workflow/scripts/report/context_phylogeny_data.R new file mode 100644 index 0000000..842942b --- /dev/null +++ b/workflow/scripts/report/context_phylogeny_data.R @@ -0,0 +1,104 @@ +#!/usr/bin/env Rscript + +# Write stdout and stderr to log file +log <- file(snakemake@log[[1]], open = "wt") +sink(log, type = "message") +sink(log, type = "output") + +library(tidyverse) +library(jsonlite) +library(ape) +library(ggtree) +library(logger) + +log_threshold(INFO) + +# Import file with plots style +source(snakemake@params[["design"]]) + +tree_ml <- read.tree(snakemake@input[["tree"]]) %>% + root( + snakemake@params[["ref_name"]], + resolve.root = TRUE + ) + +target_names <- read.dna( + snakemake@input[["target_fasta"]], + format = "fasta", + as.matrix = FALSE, +) + +target_names <- target_names[ + !startsWith(names(target_names), snakemake@config[["ALIGNMENT_REFERENCE"]]) +] +target_names <- names(target_names) + +# ML tree with context data +# Internal nodes color +# Node labels contain SH-aLRT/UFboot values +aLRT.values <- sapply( + strsplit(tree_ml$node.label, "/"), + function(x) { + as.numeric(x[1]) + } +) + +bootstrap.values <- sapply( + strsplit(tree_ml$node.label, "/"), + function(x) { + as.numeric(x[2]) + } +) + +aLRT.mask <- aLRT.values >= snakemake@params[["alrt_th"]] +boot.mask <- bootstrap.values >= snakemake@params[["boot_th"]] + +# MRCA for target samples +log_info("Calculating MRCA of target samples") +target.mrca <- getMRCA(tree_ml, target_names) +target.mrca.clade <- extract.clade(tree_ml, target.mrca) +target.mrca.clade.ntips <- Ntip(target.mrca.clade) + +log_info("Building M-L tree annotation") +tree_ml.ntips <- length(tree_ml$tip.label) +tree_ml.nodes <- tree_ml$Nnode + tree_ml.ntips +tree_ml.labels <- tree_ml$tip.label[1:tree_ml.nodes] +tree_ml.node.pass <- c(rep(FALSE, tree_ml.ntips), aLRT.mask & boot.mask) + +ml.tree.annot <- tibble( + node = 1:tree_ml.nodes, +) %>% + mutate( + Class = case_when( + tree_ml.labels %in% target_names ~ TREE_LEGEND_NAMES["tip_label"], + tree_ml.node.pass ~ TREE_LEGEND_NAMES["boot_alrt_pass"], + TRUE ~ NA + ) + ) + +# Write output files +log_info("Writing tree annotation") +write_csv(ml.tree.annot, snakemake@output$annotation) + +log_info("Writing JSON data") +target.node <- tree_ml$node.label[target.mrca - length(tree_ml$tip.label)] +list( + "boot" = strsplit(target.node, "/")[[1]][2] %>% as.numeric(), + "alrt" = strsplit(target.node, "/")[[1]][1] %>% as.numeric(), + "monophyly" = ifelse( + is.monophyletic(tree_ml, target_names), + "are", + "are not" + ), + "target_mrca" = target.mrca, + "clade_tips" = target.mrca.clade.ntips, + "max_tip_length" = max(node.depth.edgelength(tree_ml)[ + 1:length(tree_ml$tip.label) + ]), + "root" = snakemake@params[["ref_name"]] +) %>% + write_json( + snakemake@output$json, + auto_unbox = TRUE, + digits = NA + ) diff --git a/workflow/scripts/report/context_phylogeny_plot.R b/workflow/scripts/report/context_phylogeny_plot.R new file mode 100644 index 0000000..1d519a8 --- /dev/null +++ b/workflow/scripts/report/context_phylogeny_plot.R @@ -0,0 +1,61 @@ +#!/usr/bin/env Rscript + +# Write stdout and stderr to log file +log <- file(snakemake@log[[1]], open = "wt") +sink(log, type = "message") +sink(log, type = "output") + +library(tidyverse) +library(jsonlite) +library(ape) +library(ggtree) +library(logger) + +log_threshold(INFO) + +# Import file with plots style +source(snakemake@params[["design"]]) + +log_info("Reading JSON data") +json <- read_json(snakemake@input$json) + +log_info("Reading tree") +tree_ml <- read.tree(snakemake@input[["tree"]]) %>% + root( + json$root, + resolve.root = TRUE + ) + +log_info("Reading tree annotation") +annotation <- read_csv(snakemake@input$annotation) + +log_info("Plotting M-L tree with context samples") +p <- ggtree(tree_ml, layout = "circular") %<+% annotation + + geom_highlight(node = json$target_mrca, colour = "red", alpha = 0) + + geom_point(aes(color = Class, size = Class)) + + geom_treescale(1.05 * json$max_tip_length) + + geom_rootedge(0.05 * json$max_tip_length) + + xlim(-json$max_tip_length / 3, NA) + + scale_color_manual( + values = setNames( + TREE_PALETTE[names(TREE_LEGEND_NAMES)], + TREE_LEGEND_NAMES + ), + na.translate = FALSE + ) + + scale_size_manual( + values = setNames( + TREE_NODE_SIZE[names(TREE_LEGEND_NAMES)], + TREE_LEGEND_NAMES + ), + na.translate = FALSE + ) + +ggsave( + filename = snakemake@output[["plot"]], + plot = p, + width = snakemake@params[["plot_width_mm"]], + height = snakemake@params[["plot_height_mm"]], + units = "mm", + dpi = 250 +) diff --git a/workflow/scripts/report/phylo_plots.R b/workflow/scripts/report/phylo_plots.R index dc1a622..f16c312 100644 --- a/workflow/scripts/report/phylo_plots.R +++ b/workflow/scripts/report/phylo_plots.R @@ -20,25 +20,8 @@ log_threshold(INFO) # Import file with plots style source(snakemake@params[["design"]]) -# legend thresholds for ml tree -legend.names <- c( - tip_label = "Target samples", - boot_alrt_pass = sprintf( - "UFBoot ≥ %s%s & SH-aLRT ≥ %s%s ", - snakemake@params[["boot_th"]], - "%", - snakemake@params[["alrt_th"]], - "%" - ) -) - matrix <- read_csv(snakemake@input[["dist"]]) metadata <- read_csv(snakemake@input[["metadata"]]) -tree_ml <- read.tree(snakemake@input[["ml"]]) %>% - root( - snakemake@params[["ref_name"]], - resolve.root = TRUE - ) study_names <- read.dna( snakemake@input[["study_fasta"]], @@ -189,76 +172,6 @@ ggsave( dpi = 250 ) -# ML tree with context data - -# Internal nodes color -# Node labels contain SH-aLRT/UFboot values -aLRT.values <- sapply( - strsplit(tree_ml$node.label, "/"), - function(x) { - as.numeric(x[1]) - } -) - -bootstrap.values <- sapply( - strsplit(tree_ml$node.label, "/"), - function(x) { - as.numeric(x[2]) - } -) - -aLRT.mask <- aLRT.values >= snakemake@params[["alrt_th"]] -boot.mask <- bootstrap.values >= snakemake@params[["boot_th"]] - -# MRCA for target samples -log_info("Calculating MRCA of target samples") -study.mrca <- getMRCA(tree_ml, study_names) -study.mrca.clade <- extract.clade(tree_ml, study.mrca) -study.mrca.clade.ntips <- Ntip(study.mrca.clade) - -log_info("Building M-L tree annotation") -tree_ml.max.tip.length <- max(node.depth.edgelength(tree_ml)[1:length(tree$tip.label)]) -tree_ml.ntips <- length(tree_ml$tip.label) -tree_ml.nodes <- tree_ml$Nnode + tree_ml.ntips -tree_ml.labels <- tree_ml$tip.label[1:tree_ml.nodes] -tree_ml.node.pass <- c(rep(FALSE, tree_ml.ntips), aLRT.mask & boot.mask) - -ml.tree.annot <- tibble( - node = 1:tree_ml.nodes, - ) %>% - mutate( - Class = case_when( - tree_ml.labels %in% study_names ~ legend.names["tip_label"], - tree_ml.node.pass ~ legend.names["boot_alrt_pass"], - TRUE ~ NA - ) - ) - -log_info("Plotting M-L tree with context samples") -p <- ggtree(tree_ml, layout = "circular") %<+% ml.tree.annot + - geom_highlight(node = study.mrca, colour = "red", alpha = 0) + - geom_point(aes(color = Class, size = Class)) + - geom_treescale(1.05 * tree_ml.max.tip.length) + - geom_rootedge(0.05 * tree_ml.max.tip.length) + - xlim(-tree_ml.max.tip.length / 3, NA) + - scale_color_manual( - values = setNames(TREE_PALETTE[names(legend.names)], legend.names), - na.translate = FALSE - ) + - scale_size_manual( - values = setNames(TREE_NODE_SIZE[names(legend.names)], legend.names), - na.translate = FALSE - ) - -ggsave( - filename = snakemake@output[["tree_ml"]], - plot = p, - width = snakemake@params[["plot_width_mm"]], - height = snakemake@params[["plot_height_mm"]], - units = "mm", - dpi = 250 -) - # PLOT TABLES log_info("Saving temporal signal table") tempest %>% @@ -272,20 +185,13 @@ tempest %>% # TEMPEST STATS model <- lm(distance ~ date_interval, data = tempest) -p.value <- summary(model)$coefficients[2,4] +p.value <- summary(model)$coefficients[2, 4] # TREE STATS -study.node <- tree_ml$node.label[study.mrca - length(tree_ml$tip.label)] -monophyletic <- ifelse(is.monophyletic(tree_ml, study_names), "are", "are not") - list( "sub_rate" = model$coefficients[[2]] * 365, "r2" = summary(model)$r.squared[[1]], - "pvalue" = ifelse(p.value < 0.001, "< 0.001", p.value), - "boot" = strsplit(study.node, "/")[[1]][2] %>% as.numeric(), - "alrt" = strsplit(study.node, "/")[[1]][1] %>% as.numeric(), - "monophyly" = monophyletic, - "clade_tips" = study.mrca.clade.ntips + "pvalue" = ifelse(p.value < 0.001, "< 0.001", p.value) ) %>% toJSON() %>% write(snakemake@output[["json"]]) From 016d370a0863100b96af1ae149bb144695aceba2 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 2 Sep 2025 17:07:39 +0200 Subject: [PATCH 021/138] Decouple distance tree and time signal analysis plots from data --- workflow/rules/report.smk | 80 ++++--- .../scripts/report/allele_freq_tree_data.R | 54 +++++ .../scripts/report/allele_freq_tree_plot.R | 85 ++++++++ workflow/scripts/report/phylo_plots.R | 197 ------------------ workflow/scripts/report/time_signal_data.R | 67 ++++++ workflow/scripts/report/time_signal_plot.R | 47 +++++ 6 files changed, 305 insertions(+), 225 deletions(-) create mode 100644 workflow/scripts/report/allele_freq_tree_data.R create mode 100644 workflow/scripts/report/allele_freq_tree_plot.R delete mode 100644 workflow/scripts/report/phylo_plots.R create mode 100644 workflow/scripts/report/time_signal_data.R create mode 100644 workflow/scripts/report/time_signal_plot.R diff --git a/workflow/rules/report.smk b/workflow/rules/report.smk index 7e2c64d..1f96608 100644 --- a/workflow/rules/report.smk +++ b/workflow/rules/report.smk @@ -157,46 +157,70 @@ rule context_phylogeny_plot: "../scripts/report/context_phylogeny_plot.R" -# rule allele_freq_phylogeny_data: # nj -# ... - - -# rule allele_freq_phylogeny_plot: -# ... +rule allele_freq_tree_data: + conda: "../envs/renv.yaml" + params: + use_bionj = config["USE_BIONJ"], + ref_name = config["ALIGNMENT_REFERENCE"], + input: + dist = REPORT_DIR_TABLES/"distances.csv", + output: + tree = report(REPORT_DIR_TABLES/"allele_freq_tree.nwk"), + log: + LOGDIR / "allele_freq_tree_data" / "log.txt" + script: + "../scripts/report/allele_freq_tree_data.R" -# rule time_signal_data: # tempest -# ... +rule allele_freq_tree_plot: + conda: "../envs/renv.yaml" + params: + design = config["PLOTS"], + ref_name = config["ALIGNMENT_REFERENCE"], + plot_height_mm = 119.4, + plot_width_mm = 159.2, + input: + tree = report(REPORT_DIR_TABLES/"allele_freq_tree.nwk"), + study_fasta = OUTDIR/f"{OUTPUT_NAME}.fasta", + metadata = config["METADATA"], + output: + plot = report(REPORT_DIR_PLOTS/"allele_freq_tree.png"), + log: + LOGDIR / "allele_freq_tree_plot" / "log.txt" + script: + "../scripts/report/allele_freq_tree_plot.R" -# rule time_signal_plot: # tempest -# ... +rule time_signal_data: + conda: "../envs/renv.yaml" + params: + ref_name = config["ALIGNMENT_REFERENCE"], + input: + tree = report(REPORT_DIR_TABLES/"allele_freq_tree.nwk"), + metadata = config["METADATA"], + output: + table = report(REPORT_DIR_TABLES/"time_signal.csv"), + json = REPORT_DIR_TABLES/"time_signal.json" + log: + LOGDIR / "time_signal_data" / "log.txt" + script: + "../scripts/report/time_signal_data.R" -rule phylo_plots: +rule time_signal_plot: conda: "../envs/renv.yaml" params: design = config["PLOTS"], - ref_name = config["ALIGNMENT_REFERENCE"], - boot_th = 95, - alrt_th = 80, plot_height_mm = 119.4, plot_width_mm = 159.2, - use_bionj = config["USE_BIONJ"] input: - dist = REPORT_DIR_TABLES/f"distances.csv", - study_fasta = OUTDIR/f"{OUTPUT_NAME}.fasta", - ml = OUTDIR/f"tree_context/{OUTPUT_NAME}.treefile", - metadata = config["METADATA"] + table = report(REPORT_DIR_TABLES/"time_signal.csv"), output: - temest = report(REPORT_DIR_PLOTS/"figure_9.png"), - tree = report(REPORT_DIR_PLOTS/"figure_8.png"), - table = report(REPORT_DIR_TABLES/"figure_9.csv"), - json = temp(OUTDIR/"stats.lm.json") + plot = report(REPORT_DIR_PLOTS/"time_signal.png"), log: - LOGDIR / "phylo_plots" / "log.txt" + LOGDIR / "time_signal_plot" / "log.txt" script: - "../scripts/report/phylo_plots.R" + "../scripts/report/time_signal_plot.R" rule evo_plots: @@ -263,14 +287,14 @@ rule report: SNV_spike = report(REPORT_DIR_PLOTS/"figure_5b.png"), volcano = report(REPORT_DIR_PLOTS/"figure_6.png"), panel = report(REPORT_DIR_PLOTS/"figure_7.png"), - tree = report(REPORT_DIR_PLOTS/"figure_8.png"), - temest = report(REPORT_DIR_PLOTS/"figure_9.png"), + tree = report(REPORT_DIR_PLOTS/"allele_freq_tree.png"), + temest = report(REPORT_DIR_PLOTS/"time_signal.png"), heat_table = report(REPORT_DIR_TABLES/"heatmap.csv"), evo = report(REPORT_DIR_PLOTS/"figure_11.png"), omega_plot = report(REPORT_DIR_PLOTS/"figure_12.png"), freyja_ts = OUTDIR/"demixing"/"freyja_data"/"last_barcode_update.txt", value = REPORT_DIR_TABLES/"diversity.json", - stats_lm = OUTDIR/"stats.lm.json", + stats_lm = REPORT_DIR_TABLES/"time_signal.json", stats_ml = REPORT_DIR_TABLES/"context_phylogeny.json", table = OUTDIR/"summary_table.csv", sum_nv = OUTDIR/"summary_nv.json", diff --git a/workflow/scripts/report/allele_freq_tree_data.R b/workflow/scripts/report/allele_freq_tree_data.R new file mode 100644 index 0000000..f7bd7aa --- /dev/null +++ b/workflow/scripts/report/allele_freq_tree_data.R @@ -0,0 +1,54 @@ +#!/usr/bin/env Rscript + +# Write stdout and stderr to log file +log <- file(snakemake@log[[1]], open = "wt") +sink(log, type = "message") +sink(log, type = "output") + +library(tidyverse) +library(data.table) +library(ape) +library(logger) + +fix_negative_edge_length <- function(nj.tree) { + edge_infos <- cbind( + nj.tree$edge, + nj.tree$edge.length + ) %>% + as.data.table() + colnames(edge_infos) <- c("from", "to", "length") + nega_froms <- edge_infos[length < 0, sort(unique(from))] + for (nega_from in nega_froms) { + minus_length <- edge_infos[from == nega_from, ][order(length)][1, length] + edge_infos[from == nega_from, length := length - minus_length] + edge_infos[to == nega_from, length := length + minus_length] + } + nj.tree$edge.length <- edge_infos$length + nj.tree +} + +log_threshold(INFO) + +matrix <- read_csv(snakemake@input$dist) + +# Tree construction +if (snakemake@params$use_bionj) { + log_info("Constructing BIONJ tree based on distances") + tree_method <- bionj +} else { + log_info("Constructing NJ tree based on distances") + tree_method <- nj +} +tree <- matrix %>% + column_to_rownames(var = "...1") %>% + as.matrix() %>% + as.dist() %>% + tree_method() %>% + root(snakemake@params$ref_name, resolve.root = TRUE) + +# Resolve possible negative edge lengths in tree +log_info("Resolving negative edge lengths") +tree <- fix_negative_edge_length(tree) + +log_info("Saving tree") +write.tree(tree, snakemake@output$tree) diff --git a/workflow/scripts/report/allele_freq_tree_plot.R b/workflow/scripts/report/allele_freq_tree_plot.R new file mode 100644 index 0000000..5ccb603 --- /dev/null +++ b/workflow/scripts/report/allele_freq_tree_plot.R @@ -0,0 +1,85 @@ +#!/usr/bin/env Rscript + +# Write stdout and stderr to log file +log <- file(snakemake@log[[1]], open = "wt") +sink(log, type = "message") +sink(log, type = "output") + +library(tidyverse) +library(ape) +library(ggtree) +library(logger) + +log_threshold(INFO) + +# Import file with plots style +source(snakemake@params[["design"]]) + +log_info("Building tree annotation") + +# Read metadata +log_debug("Reading metadata") +metadata <- read_csv(snakemake@input$metadata) + +# Extract names from records +log_debug("Reading FASTA") +study_names <- read.dna( + snakemake@input$study_fasta, + format = "fasta", + as.matrix = FALSE, +) +log_debug("Extracting names") +study_names <- study_names[ + !startsWith(names(study_names), snakemake@params$ref_name) +] +study_names <- names(study_names) + +# Obtain sample names ordered by CollectionDate +log_debug("Sorting names by collection date") +date_order <- metadata %>% + arrange(CollectionDate) %>% + filter(ID %in% study_names) %>% + pull(ID) %>% + unique() + +log_debug("Building annotation dataframe") +tree_tiplab <- data.frame( + ID = date_order, + order = seq(1, length(date_order), 1) +) %>% + rowwise() %>% + mutate( + tip_label = sprintf( + "(%s)-%s", + order, + ID + ) + ) %>% + ungroup() %>% + add_row( + ID = snakemake@params$ref_name, + order = 0, + tip_label = snakemake@params$ref_name + ) + +# Read distance tree and root +log_info("Reading tree") +tree <- read.tree(snakemake@input$tree) %>% + root(snakemake@params$ref_name, resolve.root = TRUE) + +# Plot +log_info("Plotting distance tree") +max.tip.length <- max(node.depth.edgelength(tree)[1:length(tree$tip.label)]) +p <- ggtree(tree) %<+% tree_tiplab + + geom_tiplab(aes(label = tip_label)) + + geom_treescale(1.1 * max.tip.length) + + geom_rootedge(0.05 * max.tip.length) + +ggsave( + filename = snakemake@output$plot, + plot = p, + width = snakemake@params$plot_width_mm, + height = snakemake@params$plot_height_mm, + units = "mm", + dpi = 250 +) diff --git a/workflow/scripts/report/phylo_plots.R b/workflow/scripts/report/phylo_plots.R deleted file mode 100644 index f16c312..0000000 --- a/workflow/scripts/report/phylo_plots.R +++ /dev/null @@ -1,197 +0,0 @@ -#!/usr/bin/env Rscript - -# Write stdout and stderr to log file -log <- file(snakemake@log[[1]], open = "wt") -sink(log, type = "message") -sink(log, type = "output") - -library(tidyverse) -library(stringi) -library(ape) -library(ggtree) -library(data.table) -library(ggpubr) -library(pegas) -library(jsonlite) -library(logger) - -log_threshold(INFO) - -# Import file with plots style -source(snakemake@params[["design"]]) - -matrix <- read_csv(snakemake@input[["dist"]]) -metadata <- read_csv(snakemake@input[["metadata"]]) - -study_names <- read.dna( - snakemake@input[["study_fasta"]], - format = "fasta", - as.matrix = FALSE, -) - -study_names <- study_names[ - !startsWith(names(study_names), snakemake@config[["ALIGNMENT_REFERENCE"]]) -] -study_names <- names(study_names) - -# Obtain sample names ordered by CollectionDate -date_order <- metadata %>% - arrange(CollectionDate) %>% - filter(ID %in% study_names) %>% - pull(ID) %>% - unique() - -tree_tiplab <- data.frame( - ID = date_order, - order = seq(1, length(date_order), 1) -) %>% - rowwise() %>% - mutate( - tip_label = sprintf( - "(%s)-%s", - order, - ID - ) - ) %>% - ungroup() %>% - add_row( - ID = snakemake@params[["ref_name"]], - order = 0, - tip_label = snakemake@params[["ref_name"]] - ) - - -# DATA PROCESSING #### -# Resolve possible negative edge lengths in n-j tree -fix_negative_edge_length <- function(nj.tree) { - edge_infos <- cbind( - nj.tree$edge, - nj.tree$edge.length - ) %>% - as.data.table - colnames(edge_infos) <- c("from", "to", "length") - - nega_froms <- edge_infos[length < 0, sort(unique(from))] - - for (nega_from in nega_froms) { - minus_length <- edge_infos[from == nega_from, ][order(length)][1, length] - edge_infos[from == nega_from, length := length - minus_length] - edge_infos[to == nega_from, length := length + minus_length] - } - nj.tree$edge.length <- edge_infos$length - nj.tree -} - -# Tree construction -if (snakemake@params$use_bionj) { - log_info("Constructing BIONJ tree based on distances") - tree_method = bionj -} else { - log_info("Constructing NJ tree based on distances") - tree_method = nj -} -tree <- matrix %>% - column_to_rownames(var = "...1") %>% - as.matrix() %>% - as.dist() %>% - tree_method() %>% - root(snakemake@params[["ref_name"]], resolve.root = TRUE) - -tree <- fix_negative_edge_length(tree) - -# Get patristic distances to ancestor from n-j tree -log_info("Getting patristic distances to ancestor from n-j tree") -tempest <- adephylo::distRoot( - tree, - "all", - method = "patristic" -) %>% - as.data.frame() %>% - rownames_to_column(var = "ID") %>% - filter(ID != snakemake@params[["ref_name"]]) %>% - rename(distance = ".") %>% - left_join( - select( - metadata, - ID, - CollectionDate - ) - ) %>% - mutate( - date_interval = as.numeric( - as.Date(CollectionDate) - min(as.Date(CollectionDate)) - ) - ) - - -# PLOTS #### -# (BIO)NJ tree -log_info("Plotting distance tree") -tree_plot <- ggtree(tree) %<+% - tree_tiplab + - geom_tiplab(aes(label = tip_label)) + - geom_treescale() + - geom_rootedge(0.5) + - xlim(0, max(tempest$distance) + 3.5) - -ggsave( - filename = snakemake@output[["tree"]], - plot = tree_plot, - width = snakemake@params[["plot_width_mm"]], - height = snakemake@params[["plot_height_mm"]], - units = "mm", - dpi = 250 -) - -# TempEst -log_info("Plotting temporal signal analysis") -tempest_fig <- tempest %>% - ggplot() + - aes( - x = date_interval, - y = distance - ) + - geom_smooth( - method = "lm", - fill = "gray95", - alpha = 0.6, - color = "red" - ) + - geom_point() + - labs( - y = "Root-to-tip distance", - x = "Days since the initial sampling" - ) - -ggsave( - filename = snakemake@output[["temest"]], - plot = tempest_fig, - width = snakemake@params[["plot_width_mm"]], - height = snakemake@params[["plot_height_mm"]], - units = "mm", - dpi = 250 -) - -# PLOT TABLES -log_info("Saving temporal signal table") -tempest %>% - transmute( - sample = ID, - Distance = distance, - CollectionDate = CollectionDate, - DaysSinceFirst = date_interval - ) %>% - write.csv(snakemake@output[["table"]], row.names = FALSE) - -# TEMPEST STATS -model <- lm(distance ~ date_interval, data = tempest) -p.value <- summary(model)$coefficients[2, 4] - -# TREE STATS -list( - "sub_rate" = model$coefficients[[2]] * 365, - "r2" = summary(model)$r.squared[[1]], - "pvalue" = ifelse(p.value < 0.001, "< 0.001", p.value) -) %>% - toJSON() %>% - write(snakemake@output[["json"]]) diff --git a/workflow/scripts/report/time_signal_data.R b/workflow/scripts/report/time_signal_data.R new file mode 100644 index 0000000..b270ebf --- /dev/null +++ b/workflow/scripts/report/time_signal_data.R @@ -0,0 +1,67 @@ +#!/usr/bin/env Rscript + +# Write stdout and stderr to log file +log <- file(snakemake@log[[1]], open = "wt") +sink(log, type = "message") +sink(log, type = "output") + +library(tidyverse) +library(jsonlite) +library(ape) +library(adephylo) +library(logger) + +log_threshold(INFO) + +# Read distance tree and root +log_info("Reading tree") +tree <- read.tree(snakemake@input$tree) %>% + root(snakemake@params$ref_name, resolve.root = TRUE) + +# Read metadata +log_info("Reading metadata") +metadata <- read_csv(snakemake@input$metadata) + +# Get patristic distances to ancestor in distance tree +log_info("Calculating patristic distances to ancestor in distance tree") +time.signal <- distRoot( + tree, + "all", + method = "patristic" +) %>% + as.data.frame() %>% + rownames_to_column(var = "ID") %>% + filter(ID != snakemake@params$ref_name) %>% + rename(distance = ".") %>% + left_join( + select( + metadata, + ID, + CollectionDate + ) + ) %>% + mutate( + date_interval = as.numeric( + as.Date(CollectionDate) - min(as.Date(CollectionDate)) + ) + ) + +# Save table +log_info("Saving time signal data") +log_debug("Saving table") +write_csv(time.signal, snakemake@output$table) + +# Time signal stats +log_debug("Building linear model") +model <- lm(distance ~ date_interval, data = time.signal) +p.value <- summary(model)$coefficients[2, 4] + +# TREE STATS +log_debug("Saving linear model") +list( + "sub_rate" = model$coefficients[[2]] * 365, + "r2" = summary(model)$r.squared[[1]], + "pvalue" = ifelse(p.value < 0.001, "< 0.001", p.value) +) %>% + toJSON() %>% + write(snakemake@output$json) diff --git a/workflow/scripts/report/time_signal_plot.R b/workflow/scripts/report/time_signal_plot.R new file mode 100644 index 0000000..d13299a --- /dev/null +++ b/workflow/scripts/report/time_signal_plot.R @@ -0,0 +1,47 @@ +#!/usr/bin/env Rscript + +# Write stdout and stderr to log file +log <- file(snakemake@log[[1]], open = "wt") +sink(log, type = "message") +sink(log, type = "output") + +library(tidyverse) +library(logger) + +log_threshold(INFO) + +# Import file with plots style +source(snakemake@params[["design"]]) + +log_info("Reading time signal data") +time.signal <- read_csv(snakemake@input$table) + +# PLOTS #### +# TempEst +log_info("Plotting time signal data") +p <- time.signal %>% + ggplot() + + aes( + x = date_interval, + y = distance + ) + + geom_smooth( + method = "lm", + fill = "gray95", + alpha = 0.6, + color = "red" + ) + + geom_point() + + labs( + y = "Root-to-tip distance", + x = "Days since the initial sampling" + ) + +ggsave( + filename = snakemake@output$plot, + plot = p, + width = snakemake@params[["plot_width_mm"]], + height = snakemake@params[["plot_height_mm"]], + units = "mm", + dpi = 250 +) From 63f5cf2c40ba29b95d5e3b77341803f25f0b3bfa Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Wed, 3 Sep 2025 10:55:42 +0200 Subject: [PATCH 022/138] Extract SARS-CoV-2 feature coordinates to JSON file --- .test/targets.yaml | 2 ++ config/README.md | 1 + config/config.yaml | 2 ++ config/sarscov2_coordinates.json | 23 +++++++++++++++ workflow/Snakefile | 2 +- workflow/rules/report.smk | 1 + workflow/scripts/report/NV_description.R | 36 ++++++------------------ 7 files changed, 38 insertions(+), 29 deletions(-) create mode 100644 config/sarscov2_coordinates.json diff --git a/.test/targets.yaml b/.test/targets.yaml index b9e4784..73c05fb 100644 --- a/.test/targets.yaml +++ b/.test/targets.yaml @@ -42,5 +42,7 @@ REPORT_QMD: "template.qmd" FEATURES_JSON: "config/sarscov2_features.json" +COORDINATES_JSON: + "config/sarscov2_coordinates.json" GENETIC_CODE_JSON: "config/standard_genetic_code.json" diff --git a/config/README.md b/config/README.md index 2bd06d5..1e97134 100644 --- a/config/README.md +++ b/config/README.md @@ -131,6 +131,7 @@ All of the following variables are pre-defined in [config.yaml](/config/config.y - `ALIGNMENT_REFERENCE`: NCBI accession number of the reference record for sequence alignment. - `PROBLEMATIC_VCF`: URL or path of a VCF file containing problematic genome positions for masking. - `FEATURES_JSON`: path of a JSON file containing name equivalences of genome features for data visualization. +- `COORDINATES_JSON`: path of a JSON file containing genome feature coordinates for data visualization. - `GENETIC_CODE_JSON`: path of a JSON file containing a genetic code for gene translation. - `TREE_MODEL`: substitution model used by IQTREE (see [docs](http://www.iqtree.org/doc/Substitution-Models)). - `UFBOOT_REPS`: ultrafast bootstrap replicates for IQTREE (see [UFBoot](https://doi.org/10.1093/molbev/msx281)). diff --git a/config/config.yaml b/config/config.yaml index d3ef082..63f56e3 100644 --- a/config/config.yaml +++ b/config/config.yaml @@ -4,6 +4,8 @@ PROBLEMATIC_VCF: "https://raw.githubusercontent.com/W-L/ProblematicSites_SARS-CoV2/da322c32004f7b16bdaa6a8ee7fd24d56e79d9dc/problematic_sites_sarsCov2.vcf" FEATURES_JSON: "config/sarscov2_features.json" +COORDINATES_JSON: + "config/sarscov2_coordinates.json" GENETIC_CODE_JSON: "config/standard_genetic_code.json" TREE_MODEL: diff --git a/config/sarscov2_coordinates.json b/config/sarscov2_coordinates.json new file mode 100644 index 0000000..9626dce --- /dev/null +++ b/config/sarscov2_coordinates.json @@ -0,0 +1,23 @@ +{ + "five_prime_UTR": [1, 265], + "orf1ab": [266, 21555], + "Intergenic_1": [21556, 21562], + "S": [21563, 25384], + "Intergenic_2": [25385, 25392], + "ORF3a": [25393, 26220], + "Intergenic_3": [26221, 26244], + "E": [26245, 26472], + "Intergenic_4": [26473, 26522], + "M": [26523, 27191], + "Intergenic_5": [27192, 27201], + "ORF6": [27202, 27387], + "Intergenic_6": [27388, 27393], + "ORF7": [27394, 27759], + "Intergenic_7": [27760, 27893], + "ORF8": [27894, 28259], + "Intergenic_8": [28260, 28273], + "N": [28274, 29533], + "Intergenic_9": [29534, 29557], + "ORF10": [29558, 29674], + "three_prime_UTR": [29675, 29903] +} diff --git a/workflow/Snakefile b/workflow/Snakefile index e8456e1..97381f5 100644 --- a/workflow/Snakefile +++ b/workflow/Snakefile @@ -6,7 +6,7 @@ import subprocess min_version("7.19") -__version__ = "1.2.2" +__version__ = "1.3.0" containerized: "docker://ahmig/vipera:v" + __version__ diff --git a/workflow/rules/report.smk b/workflow/rules/report.smk index 1f96608..641e0eb 100644 --- a/workflow/rules/report.smk +++ b/workflow/rules/report.smk @@ -99,6 +99,7 @@ rule general_NV_description: samples = expand("{sample}", sample = iter_samples()), design = config["PLOTS"], regions = config["PLOT_GENOME_REGIONS"], + coordinates = config["COORDINATES_JSON"], window = config["WINDOW"]["WIDTH"], step = config["WINDOW"]["STEP"], max_alt_freq = 1.0 - config["VC"]["IVAR_FREQ"] diff --git a/workflow/scripts/report/NV_description.R b/workflow/scripts/report/NV_description.R index 210e2f4..03529dd 100644 --- a/workflow/scripts/report/NV_description.R +++ b/workflow/scripts/report/NV_description.R @@ -16,30 +16,10 @@ log_threshold(INFO) # Import file with plots style source(snakemake@params[["design"]]) -# SARS-CoV-2 anotation for genome scheme -SCov2_annotation <- list( - "five_prime_UTR" = c(1:265), - "orf1ab" = c(266:21555), - "Intergenic_1" = c(21556:21562), - "S" = c(21563:25384), - "Intergenic_2" = c(25385:25392), - "ORF3a" = c(25393:26220), - "Intergenic_3" = c(26221:26244), - "E" = c(26245:26472), - "Intergenic_4" = c(26473:26522), - "M" = c(26523:27191), - "Intergenic_5" = c(27192:27201), - "ORF6" = c(27202:27387), - "Intergenic_6" = c(27388:27393), - "ORF7" = c(27394:27759), - "Intergenic_7" = c(27760:27893), - "ORF8" = c(27894:28259), - "Intergenic_8" = c(28260:28273), - "N" = c(28274:29533), - "Intergenic_9" = c(29534:29557), - "ORF10" = c(29558:29674), - "three_prime_UTR" = c(29675:29903) -) +log_info("Reading inputs") + +# Anotation for genome scheme +coordinates <- read_json(snakemake@input$coordinates) vcf <- read_delim(snakemake@input[["vcf"]]) vcf_snp <- vcf @@ -78,7 +58,7 @@ vcf <- vcf %>% ALT ) -# Df with gene length for scheme +# Create dataframe with gene length for scheme notation_empty <- data.frame( gene = "", len = 0 @@ -86,12 +66,12 @@ notation_empty <- data.frame( filter(len != 0) notation <- lapply( - names(SCov2_annotation), + names(coordinates), function(x) { add_row( notation_empty, gene = x, - len = length(SCov2_annotation[[x]]) + len = length(coordinates[[x]]) ) } ) %>% @@ -184,7 +164,7 @@ variants <- vcf %>% aes( x = len, y = 0.3, - fill = factor(gene, rev(names(SCov2_annotation))) + fill = factor(gene, rev(names(coordinates))) ), inherit.aes = FALSE, width = 0.3 From 7a16b477035da8a83901158a5aaab928a744d3c3 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Wed, 3 Sep 2025 10:57:39 +0200 Subject: [PATCH 023/138] Move inputs to appropriate directive --- workflow/rules/report.smk | 4 ++-- workflow/scripts/report/NV_description.R | 2 +- 2 files changed, 3 insertions(+), 3 deletions(-) diff --git a/workflow/rules/report.smk b/workflow/rules/report.smk index 641e0eb..2290f3a 100644 --- a/workflow/rules/report.smk +++ b/workflow/rules/report.smk @@ -98,12 +98,12 @@ rule general_NV_description: params: samples = expand("{sample}", sample = iter_samples()), design = config["PLOTS"], - regions = config["PLOT_GENOME_REGIONS"], - coordinates = config["COORDINATES_JSON"], window = config["WINDOW"]["WIDTH"], step = config["WINDOW"]["STEP"], max_alt_freq = 1.0 - config["VC"]["IVAR_FREQ"] input: + coordinates = config["COORDINATES_JSON"], + regions = config["PLOT_GENOME_REGIONS"], window = OUTDIR/f"{OUTPUT_NAME}.window.csv", vcf = OUTDIR/f"{OUTPUT_NAME}.masked.filtered.tsv", metadata = config["METADATA"] diff --git a/workflow/scripts/report/NV_description.R b/workflow/scripts/report/NV_description.R index 03529dd..760e6b9 100644 --- a/workflow/scripts/report/NV_description.R +++ b/workflow/scripts/report/NV_description.R @@ -118,7 +118,7 @@ vcf <- vcf %>% ) # NSP data -npc <- read_csv(snakemake@params[["regions"]]) %>% +npc <- read_csv(snakemake@input[["regions"]]) %>% mutate( summary_nsp = case_when( region %in% paste("nsp", seq(4, 12, 1), sep = "") ~ "nsp4-12", From 91ed2575bdcf87e6d5e10b7f8bebb813aae15015 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Wed, 3 Sep 2025 17:36:58 +0200 Subject: [PATCH 024/138] Fix reading of JSON coordinates --- workflow/scripts/report/NV_description.R | 7 +++++-- 1 file changed, 5 insertions(+), 2 deletions(-) diff --git a/workflow/scripts/report/NV_description.R b/workflow/scripts/report/NV_description.R index 760e6b9..9f7a0cf 100644 --- a/workflow/scripts/report/NV_description.R +++ b/workflow/scripts/report/NV_description.R @@ -19,7 +19,10 @@ source(snakemake@params[["design"]]) log_info("Reading inputs") # Anotation for genome scheme -coordinates <- read_json(snakemake@input$coordinates) +coordinates <- lapply( + read_json(snakemake@input$coordinates, simplifyVector = TRUE), + function(x) x[1]:x[2] +) vcf <- read_delim(snakemake@input[["vcf"]]) vcf_snp <- vcf @@ -468,7 +471,7 @@ model <- lm(n ~ CollectionDate, data = figur_SNP_table) # Calculate correlation, if possible if (nrow(figur_SNP_table) > 2) { - p.value <- summary(model)$coefficients[2,4] + p.value <- summary(model)$coefficients[2, 4] } else { p.value <- NA } From 82f5ab0163122ffa4a06076524fc3d5d6372a456 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Fri, 5 Sep 2025 12:16:28 +0200 Subject: [PATCH 025/138] Extract filters to params --- workflow/rules/vaf.smk | 4 ++++ workflow/scripts/filter_tsv.R | 14 ++++++-------- 2 files changed, 10 insertions(+), 8 deletions(-) diff --git a/workflow/rules/vaf.smk b/workflow/rules/vaf.smk index bd16219..99ed980 100644 --- a/workflow/rules/vaf.smk +++ b/workflow/rules/vaf.smk @@ -109,6 +109,10 @@ rule mask_tsv: rule filter_tsv: threads: 1 conda: "../envs/renv.yaml" + params: + min_depth = 20, + min_alt_rv = 2, + min_alt_dp = 2, input: tsv = OUTDIR/f"{OUTPUT_NAME}.masked.tsv", annotation = OUTDIR/"annotation.csv" diff --git a/workflow/scripts/filter_tsv.R b/workflow/scripts/filter_tsv.R index 55dc216..aae4631 100644 --- a/workflow/scripts/filter_tsv.R +++ b/workflow/scripts/filter_tsv.R @@ -12,24 +12,22 @@ log_threshold(INFO) # Read inputs data <- read_tsv(snakemake@input[["tsv"]]) -# Filtering criteria: -# - P-value < 0.05 -# - Depth >= 20 -# - For SNP more than 2 reads in each strand +# Filtering is.deletion <- str_detect( data$ALT, "^[A-Z]", negate = TRUE ) -inBothStrands <- data$ALT_RV > 2 & data$ALT_DP > 2 -Depth <- (data$ALT_RV + data$ALT_DP) >= 20 +strand.mask <- data$ALT_RV > snakemake@params$min_alt_rv & + data$ALT_DP > snakemake@params$min_alt_dp +depth.mask <- (data$ALT_RV + data$ALT_DP) >= snakemake@params$min_depth log_info("Filtering variants") data <- filter( data, as.logical(PASS), - Depth, - inBothStrands | is.deletion + depth.mask, + strand.mask | is.deletion ) log_info("Finding synonymous and non synonymous variants") From fd3e810f79499f5366622401be70b96d6f22e66a Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Fri, 5 Sep 2025 12:17:33 +0200 Subject: [PATCH 026/138] Extract variant annotation using SnpSift --- workflow/rules/vaf.smk | 20 ++++++++++++++++++++ 1 file changed, 20 insertions(+) diff --git a/workflow/rules/vaf.smk b/workflow/rules/vaf.smk index 99ed980..3ca257e 100644 --- a/workflow/rules/vaf.smk +++ b/workflow/rules/vaf.smk @@ -137,6 +137,26 @@ rule tsv_to_vcf: "../scripts/tsv_to_vcf.py" +rule extract_vcf_fields: + threads: 1 + conda: "../envs/snpeff.yaml" + params: + extract_columns = [ + "CHROM", "REF", "POS", "ALT", "DP", + '"GEN[*].ALT_DP"', '"GEN[*].ALT_RV"', '"GEN[*].ALT_FREQ"', + '"GEN[*].ALT_QUAL"', '"ANN[*].GENE"', '"ANN[*].HGVS_P"' + ], + sep = "," + input: + vcf = OUTDIR/f"{OUTPUT_NAME}.vcf" + output: + tsv = OUTDIR/f"{OUTPUT_NAME}.vcf_fields.tsv" + log: + LOGDIR / "tsv_to_vcf" / "log.txt" + shell: + 'SnpSift extractFields -s {params.sep:q} {input.vcf:q} {params.extract_columns} >{output.tsv:q} 2>{log:q}' + + rule variants_effect: threads: 1 shadow: "minimal" From 834a73f0b686855036598e5c167a66e8096c6616 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Fri, 5 Sep 2025 12:39:00 +0200 Subject: [PATCH 027/138] Add dependency --- workflow/envs/snpeff.yaml | 1 + 1 file changed, 1 insertion(+) diff --git a/workflow/envs/snpeff.yaml b/workflow/envs/snpeff.yaml index 1423a3c..612ab76 100644 --- a/workflow/envs/snpeff.yaml +++ b/workflow/envs/snpeff.yaml @@ -2,3 +2,4 @@ channels: - bioconda dependencies: - snpeff==5.1d + - snpsift==5.1d From e3bee0066dae0ec297d42fb0ab7efb0a61baffc5 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Fri, 5 Sep 2025 13:04:48 +0200 Subject: [PATCH 028/138] Fix input VCF --- workflow/rules/vaf.smk | 40 ++++++++++++++++++++-------------------- 1 file changed, 20 insertions(+), 20 deletions(-) diff --git a/workflow/rules/vaf.smk b/workflow/rules/vaf.smk index 3ca257e..6e1b958 100644 --- a/workflow/rules/vaf.smk +++ b/workflow/rules/vaf.smk @@ -137,26 +137,6 @@ rule tsv_to_vcf: "../scripts/tsv_to_vcf.py" -rule extract_vcf_fields: - threads: 1 - conda: "../envs/snpeff.yaml" - params: - extract_columns = [ - "CHROM", "REF", "POS", "ALT", "DP", - '"GEN[*].ALT_DP"', '"GEN[*].ALT_RV"', '"GEN[*].ALT_FREQ"', - '"GEN[*].ALT_QUAL"', '"ANN[*].GENE"', '"ANN[*].HGVS_P"' - ], - sep = "," - input: - vcf = OUTDIR/f"{OUTPUT_NAME}.vcf" - output: - tsv = OUTDIR/f"{OUTPUT_NAME}.vcf_fields.tsv" - log: - LOGDIR / "tsv_to_vcf" / "log.txt" - shell: - 'SnpSift extractFields -s {params.sep:q} {input.vcf:q} {params.extract_columns} >{output.tsv:q} 2>{log:q}' - - rule variants_effect: threads: 1 shadow: "minimal" @@ -187,6 +167,26 @@ rule variants_effect: """ +rule extract_vcf_fields: + threads: 1 + conda: "../envs/snpeff.yaml" + params: + extract_columns = [ + "CHROM", "POS", "REF", "ALT", + '"ANN[*].IMPACT"', '"ANN[*].BIOTYPE"', + '"ANN[*].GENE"', '"ANN[*].GENEID"', '"ANN[*].FEATURE"', '"ANN[*].HGVS_P"', '"ANN[*].HGVS_C"' + ], + sep = "," + input: + vcf = OUTDIR/f"{OUTPUT_NAME}.annotated.vcf" + output: + tsv = OUTDIR/f"{OUTPUT_NAME}.vcf_fields.tsv" + log: + LOGDIR / "tsv_to_vcf" / "log.txt" + shell: + 'SnpSift extractFields -s {params.sep:q} {input.vcf:q} {params.extract_columns} >{output.tsv:q} 2>{log:q}' + + rule vcf_to_tsv: threads: 1 conda: "../envs/renv.yaml" From 8de29809141a2bc308208dcf71b6457ace1ae673 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Fri, 5 Sep 2025 13:21:13 +0200 Subject: [PATCH 029/138] Obtain the extracted VCF fields in longer format --- workflow/rules/vaf.smk | 12 ++++++++++++ workflow/scripts/format_vcf_fields_longer.R | 12 ++++++++++++ 2 files changed, 24 insertions(+) create mode 100644 workflow/scripts/format_vcf_fields_longer.R diff --git a/workflow/rules/vaf.smk b/workflow/rules/vaf.smk index 6e1b958..c823fea 100644 --- a/workflow/rules/vaf.smk +++ b/workflow/rules/vaf.smk @@ -187,6 +187,18 @@ rule extract_vcf_fields: 'SnpSift extractFields -s {params.sep:q} {input.vcf:q} {params.extract_columns} >{output.tsv:q} 2>{log:q}' +rule format_vcf_fields_longer: + conda: "../envs/renv.yaml" + params: + sep = "," + input: + tsv = OUTDIR/f"{OUTPUT_NAME}.vcf_fields.tsv" + output: + tsv = OUTDIR/f"{OUTPUT_NAME}.vcf_fields.longer.tsv" + script: + "../scripts/format_vcf_fields_longer.R" + + rule vcf_to_tsv: threads: 1 conda: "../envs/renv.yaml" diff --git a/workflow/scripts/format_vcf_fields_longer.R b/workflow/scripts/format_vcf_fields_longer.R new file mode 100644 index 0000000..fbe314d --- /dev/null +++ b/workflow/scripts/format_vcf_fields_longer.R @@ -0,0 +1,12 @@ +#!/usr/bin/env Rscript + +# Write stdout and stderr to log file +log <- file(snakemake@log[[1]], open = "wt") +sink(log, type = "message") +sink(log, type = "output") + +library(tidyverse) + +read_tsv(snakemake@input$tsv) %>% + separate_rows(contains("[*]"), sep = snakemake@params$sep) %>% + write_tsv(snakemake@output$tsv) From e7b68c323e336d8993af4937540ab3275aefbca9 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Fri, 5 Sep 2025 13:22:02 +0200 Subject: [PATCH 030/138] Add missing log --- workflow/rules/vaf.smk | 2 ++ 1 file changed, 2 insertions(+) diff --git a/workflow/rules/vaf.smk b/workflow/rules/vaf.smk index c823fea..ab348a9 100644 --- a/workflow/rules/vaf.smk +++ b/workflow/rules/vaf.smk @@ -195,6 +195,8 @@ rule format_vcf_fields_longer: tsv = OUTDIR/f"{OUTPUT_NAME}.vcf_fields.tsv" output: tsv = OUTDIR/f"{OUTPUT_NAME}.vcf_fields.longer.tsv" + log: + LOGDIR / "format_vcf_fields_longer" / "log.txt" script: "../scripts/format_vcf_fields_longer.R" From 4a892a20d135d8e082516611340af1253bbfb596 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Fri, 5 Sep 2025 13:26:52 +0200 Subject: [PATCH 031/138] Add VCF field --- workflow/rules/vaf.smk | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/workflow/rules/vaf.smk b/workflow/rules/vaf.smk index ab348a9..62ee512 100644 --- a/workflow/rules/vaf.smk +++ b/workflow/rules/vaf.smk @@ -173,7 +173,7 @@ rule extract_vcf_fields: params: extract_columns = [ "CHROM", "POS", "REF", "ALT", - '"ANN[*].IMPACT"', '"ANN[*].BIOTYPE"', + '"ANN[*].EFFECT"', '"ANN[*].IMPACT"', '"ANN[*].BIOTYPE"', '"ANN[*].GENE"', '"ANN[*].GENEID"', '"ANN[*].FEATURE"', '"ANN[*].HGVS_P"', '"ANN[*].HGVS_C"' ], sep = "," From 52e95ea38ada383e0e7c60b8f383c9e74d73ef7f Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Fri, 5 Sep 2025 13:40:33 +0200 Subject: [PATCH 032/138] Avoid hard-codig reference name --- workflow/rules/vaf.smk | 2 ++ workflow/scripts/tsv_to_vcf.py | 2 +- 2 files changed, 3 insertions(+), 1 deletion(-) diff --git a/workflow/rules/vaf.smk b/workflow/rules/vaf.smk index 62ee512..0d58337 100644 --- a/workflow/rules/vaf.smk +++ b/workflow/rules/vaf.smk @@ -127,6 +127,8 @@ rule filter_tsv: rule tsv_to_vcf: threads: 1 conda: "../envs/biopython.yaml" + params: + ref_name = config["ALIGNMENT_REFERENCE"], input: tsv = OUTDIR/f"{OUTPUT_NAME}.masked.prefiltered.tsv", output: diff --git a/workflow/scripts/tsv_to_vcf.py b/workflow/scripts/tsv_to_vcf.py index be43d9a..2201582 100644 --- a/workflow/scripts/tsv_to_vcf.py +++ b/workflow/scripts/tsv_to_vcf.py @@ -17,7 +17,7 @@ def tsv_to_vcf(tsv_file, vcf_file): # Process each row in the TSV file for index, row in tsv_df.iterrows(): # Extract fields from the TSV row - chrom = 'NC_045512.2' + chrom = snakemake.params.ref_name pos = row['POS'] ref = row['REF'] alt = row['ALT'] From 5c040f2969db284c2304db64a039c458b5e33cab Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Fri, 5 Sep 2025 14:26:04 +0200 Subject: [PATCH 033/138] Process sample variants in parallel Also renames the REGION column to SAMPLE, keeping the intended use of REGION in a VCF, and GFF_FEATURE to GB_FEATURE, keeping the intended use of GFF_FEATURE as an output of iVar --- workflow/rules/vaf.smk | 80 ++++++++++-------------- workflow/scripts/compile_variants.R | 23 +++++++ workflow/scripts/filter_tsv.R | 3 +- workflow/scripts/report/NV_description.R | 36 ++++++----- workflow/scripts/report/evo_plots.R | 15 ++--- workflow/scripts/report/heatmap.R | 6 +- workflow/scripts/report/snp_plots.R | 11 ++-- workflow/scripts/vcf_to_tsv.R | 4 +- workflow/scripts/weighted_distances.py | 4 +- 9 files changed, 98 insertions(+), 84 deletions(-) create mode 100644 workflow/scripts/compile_variants.R diff --git a/workflow/rules/vaf.smk b/workflow/rules/vaf.smk index 0d58337..b06c76c 100644 --- a/workflow/rules/vaf.smk +++ b/workflow/rules/vaf.smk @@ -48,8 +48,6 @@ rule snps_to_ancestor: -m {params.ivar_depth} \ -g {input.gff} \ -r renamed_reference.fasta - - sed 's/'$ref'/'{wildcards.sample}'/g' {wildcards.sample}.tsv | cat > {output.tsv} """ @@ -69,39 +67,18 @@ rule annotation: "../scripts/report/get_annotation.py" -rule format_tsv: - threads:1 - shadow: "shallow" - input: - expand(OUTDIR/"{sample}.tsv", sample = iter_samples()) - output: - tsv = OUTDIR/f"{OUTPUT_NAME}.tsv" - log: - LOGDIR / "format_tsv" / "log.txt" - shell: - """ - path=`echo {input} | awk '{{print $1}}'` - grep "^REGION" "$path" > header - for tsv in {input}; do - tail -n +2 "$tsv" >> body - done - cat header body > "{output.tsv}" - rm header - """ - - rule mask_tsv: threads: 1 conda: "../envs/biopython.yaml" params: mask_class = ["mask"] input: - tsv = OUTDIR/f"{OUTPUT_NAME}.tsv", + tsv = OUTDIR/"{sample}.tsv", vcf = lambda wildcards: select_problematic_vcf() output: - masked_tsv = temp(OUTDIR/f"{OUTPUT_NAME}.masked.tsv") + masked_tsv = temp(OUTDIR/"{sample}.masked.tsv") log: - LOGDIR / "mask_tsv" / "log.txt" + LOGDIR / "mask_tsv" / "{sample}.log.txt" script: "../scripts/mask_tsv.py" @@ -114,12 +91,12 @@ rule filter_tsv: min_alt_rv = 2, min_alt_dp = 2, input: - tsv = OUTDIR/f"{OUTPUT_NAME}.masked.tsv", + tsv = OUTDIR/"{sample}.masked.tsv", annotation = OUTDIR/"annotation.csv" output: - filtered_tsv = temp(OUTDIR/f"{OUTPUT_NAME}.masked.prefiltered.tsv") + filtered_tsv = temp(OUTDIR/"{sample}.masked.prefiltered.tsv") log: - LOGDIR / "filter_tsv" / "log.txt" + LOGDIR / "filter_tsv" / "{sample}.log.txt" script: "../scripts/filter_tsv.R" @@ -130,11 +107,11 @@ rule tsv_to_vcf: params: ref_name = config["ALIGNMENT_REFERENCE"], input: - tsv = OUTDIR/f"{OUTPUT_NAME}.masked.prefiltered.tsv", + tsv = OUTDIR/"{sample}.masked.prefiltered.tsv", output: - vcf = temp(OUTDIR/f"{OUTPUT_NAME}.vcf") + vcf = temp(OUTDIR/"{sample}.vcf") log: - LOGDIR / "tsv_to_vcf" / "log.txt" + LOGDIR / "tsv_to_vcf" / "{sample}.log.txt" script: "../scripts/tsv_to_vcf.py" @@ -147,11 +124,11 @@ rule variants_effect: ref_name = config["ALIGNMENT_REFERENCE"], snpeff_data_dir = (BASE_PATH / "config" / "snpeff").resolve() input: - vcf = OUTDIR/f"{OUTPUT_NAME}.vcf" + vcf = OUTDIR/"{sample}.vcf" output: - ann_vcf = temp(OUTDIR/f"{OUTPUT_NAME}.annotated.vcf") + ann_vcf = OUTDIR/"{sample}.annotated.vcf" log: - LOGDIR / "variants_effect" / "log.txt" + LOGDIR / "variants_effect" / "{sample}.log.txt" retries: 2 shell: """ @@ -165,7 +142,7 @@ rule variants_effect: echo "Local database not found at '{params.snpeff_data_dir}', downloading from repository" fi - snpEff eff -dataDir {params.snpeff_data_dir} -noStats {params.ref_name} {input.vcf} > {output.ann_vcf} + snpEff eff -dataDir {params.snpeff_data_dir} -noStats {params.ref_name} {input.vcf} >{output.ann_vcf} """ @@ -180,11 +157,11 @@ rule extract_vcf_fields: ], sep = "," input: - vcf = OUTDIR/f"{OUTPUT_NAME}.annotated.vcf" + vcf = OUTDIR/"{sample}.annotated.vcf" output: - tsv = OUTDIR/f"{OUTPUT_NAME}.vcf_fields.tsv" + tsv = OUTDIR/"{sample}.vcf_fields.tsv" log: - LOGDIR / "tsv_to_vcf" / "log.txt" + LOGDIR / "tsv_to_vcf" / "{sample}.log.txt" shell: 'SnpSift extractFields -s {params.sep:q} {input.vcf:q} {params.extract_columns} >{output.tsv:q} 2>{log:q}' @@ -194,11 +171,11 @@ rule format_vcf_fields_longer: params: sep = "," input: - tsv = OUTDIR/f"{OUTPUT_NAME}.vcf_fields.tsv" + tsv = OUTDIR/"{sample}.vcf_fields.tsv" output: - tsv = OUTDIR/f"{OUTPUT_NAME}.vcf_fields.longer.tsv" + tsv = OUTDIR/"{sample}.vcf_fields.longer.tsv" log: - LOGDIR / "format_vcf_fields_longer" / "log.txt" + LOGDIR / "format_vcf_fields_longer" / "{sample}.log.txt" script: "../scripts/format_vcf_fields_longer.R" @@ -207,11 +184,22 @@ rule vcf_to_tsv: threads: 1 conda: "../envs/renv.yaml" input: - ann_vcf = OUTDIR/f"{OUTPUT_NAME}.annotated.vcf", - pre_tsv = OUTDIR/f"{OUTPUT_NAME}.masked.prefiltered.tsv" + ann_vcf = OUTDIR/"{sample}.annotated.vcf", + pre_tsv = OUTDIR/"{sample}.masked.prefiltered.tsv" output: - tsv = OUTDIR/f"{OUTPUT_NAME}.masked.filtered.tsv" + tsv = OUTDIR/"{sample}.masked.filtered.tsv" log: - LOGDIR / "vcf_to_tsv" / "log.txt" + LOGDIR / "vcf_to_tsv" / "{sample}.log.txt" script: "../scripts/vcf_to_tsv.R" + + +rule compile_variants: + threads: 1 + input: expand(OUTDIR/"{sample}.masked.filtered.tsv", sample=iter_samples()) + output: + tsv = OUTDIR/f"{OUTPUT_NAME}.masked.filtered.tsv" + log: + LOGDIR / "compile_variants" / "log.txt" + script: + "../scripts/compile_variants.R" diff --git a/workflow/scripts/compile_variants.R b/workflow/scripts/compile_variants.R new file mode 100644 index 0000000..d85654e --- /dev/null +++ b/workflow/scripts/compile_variants.R @@ -0,0 +1,23 @@ +#!/usr/bin/env Rscript + +# Write stdout and stderr to log file +log <- file(snakemake@log[[1]], open = "wt") +sink(log, type = "message") +sink(log, type = "output") + +library(tidyverse) +library(logger) +log_threshold(INFO) + +log_info("Reading input partial variant tables and adding sample column") +tables <- lapply( + snakemake@input, + function(path) { + read_tsv(path) %>% + mutate(SAMPLE = sub('\\.tsv$', '', path)) + } +) + +log_info("Binding and writing table") +bind_rows(tables) %>% + write_tsv(snakemake@output$tsv) diff --git a/workflow/scripts/filter_tsv.R b/workflow/scripts/filter_tsv.R index aae4631..d190bce 100644 --- a/workflow/scripts/filter_tsv.R +++ b/workflow/scripts/filter_tsv.R @@ -47,9 +47,8 @@ data <- distinct(data, pick(!GFF_FEATURE), .keep_all = TRUE) features <- read_csv(snakemake@input[["annotation"]]) data <- data %>% - select(!GFF_FEATURE) %>% left_join(features) %>% - rename(GFF_FEATURE = GEN) + rename(GB_FEATURE = GEN) log_info("Saving results") write_tsv( diff --git a/workflow/scripts/report/NV_description.R b/workflow/scripts/report/NV_description.R index 9f7a0cf..5c4b398 100644 --- a/workflow/scripts/report/NV_description.R +++ b/workflow/scripts/report/NV_description.R @@ -38,10 +38,11 @@ date_order <- metadata %>% unique() empty_vcf <- tibble( - REGION = date_order, + SAMPLE = date_order, + REGION = as.character(NA), variant = as.character(NA), ALT_FREQ = as.numeric(NA), - GFF_FEATURE = as.character(NA), + GB_FEATURE = as.character(NA), synonimous = as.character(NA), POS = as.numeric(NA), ALT = as.character(NA), @@ -52,10 +53,11 @@ empty_vcf <- tibble( # Create SNP variable and select useful variables vcf <- vcf %>% dplyr::select( - variant, + SAMPLE, REGION, + variant, ALT_FREQ, - GFF_FEATURE, + GB_FEATURE, synonimous, POS, ALT @@ -91,7 +93,7 @@ vcf <- vcf %>% TRUE ~ "SNP" ), Class = case_when( - GFF_FEATURE == "Intergenic" ~ "Intergenic", + GB_FEATURE == "Intergenic" ~ "Intergenic", TRUE ~ synonimous ), POS = as.numeric(POS) @@ -102,7 +104,7 @@ vcf <- vcf %>% NV_class == "INDEL" ~ str_length(ALT) - 1 ), indel_class = case_when( - GFF_FEATURE == "Intergenic" ~ "Intergenic", + GB_FEATURE == "Intergenic" ~ "Intergenic", NV_class == "INDEL" & indel_len %% 3 == 0 ~ "In frame", @@ -114,7 +116,7 @@ vcf <- vcf %>% ungroup() %>% mutate( group = case_when( - GFF_FEATURE == "Intergenic" ~ "Intergenic", + GB_FEATURE == "Intergenic" ~ "Intergenic", NV_class == "SNP" ~ Class, NV_class == "INDEL" ~ indel_class ) @@ -156,7 +158,7 @@ variants <- vcf %>% ggplot() + aes( x = POS, - y = factor(REGION, date_order), + y = factor(SAMPLE, date_order), shape = factor(NV_class, c("SNP", "INDEL")), color = group, alpha = ALT_FREQ @@ -312,7 +314,7 @@ variants_spike <- vcf_spike %>% ggplot() + aes( x = POS, - y = factor(REGION, date_order), + y = factor(SAMPLE, date_order), shape = factor(NV_class, c("SNP", "INDEL")), color = group, alpha = ALT_FREQ @@ -363,9 +365,9 @@ figur_SNP_table <- vcf_snp %>% filter(ALT_FREQ <= snakemake@params$max_alt_freq) %>% left_join( metadata, - by = c("REGION" = "ID") + by = c("SAMPLE" = "ID") ) %>% - group_by(REGION) %>% + group_by(SAMPLE) %>% summarise( CollectionDate = min(as.Date(CollectionDate)), n = n_distinct(POS) @@ -410,7 +412,7 @@ log_info("Saving plot tables") # Variants summary table vcf %>% select( - REGION, + SAMPLE, POS, variant, ALT_FREQ, @@ -418,7 +420,7 @@ vcf %>% group ) %>% rename( - sample = REGION, + sample = SAMPLE, Variant = variant, Class = group ) %>% @@ -444,18 +446,18 @@ window %>% # Heterozygous sites per sample table vcf_snp %>% filter(ALT_FREQ <= snakemake@params$max_alt_freq) %>% - select(!GFF_FEATURE) %>% + select(!GB_FEATURE) %>% left_join( metadata, - by = c("REGION" = "ID") + by = c("SAMPLE" = "ID") ) %>% - group_by(REGION) %>% + group_by(SAMPLE) %>% summarise( CollectionDate = min(as.Date(CollectionDate)), n_PolymorphicSites = n_distinct(POS) ) %>% ungroup() %>% - rename(sample = REGION) %>% + rename(sample = SAMPLE) %>% unique() %>% write.csv(snakemake@output[["table_3"]], row.names = FALSE) diff --git a/workflow/scripts/report/evo_plots.R b/workflow/scripts/report/evo_plots.R index 31da73b..586328e 100644 --- a/workflow/scripts/report/evo_plots.R +++ b/workflow/scripts/report/evo_plots.R @@ -21,10 +21,11 @@ N_S_position <- read_delim(snakemake@input[["N_S"]]) # Create SNP variable and select useful variables vcf <- vcf %>% dplyr::select( + SAMPLE, variant, REGION, ALT_FREQ, - GFF_FEATURE, + GB_FEATURE, synonimous, POS ) @@ -37,14 +38,14 @@ metadata <- metadata %>% ) ) %>% select(ID, interval) %>% - rename(REGION = ID) + rename(SAMPLE = ID) vcf <- left_join(vcf, metadata) # PLOT log_info("Ploting dN and dS over time") plot_df <- vcf %>% - group_by(REGION, synonimous) %>% + group_by(SAMPLE, synonimous) %>% summarise( Freq = sum(ALT_FREQ, na.rm = TRUE) ) %>% @@ -67,7 +68,7 @@ plot_df <- vcf %>% values_to = "value", names_to = "d" ) %>% - left_join(unique(select(vcf, REGION, interval))) + left_join(unique(select(vcf, SAMPLE, interval))) plot <- plot_df %>% filter(d != "w") %>% @@ -134,7 +135,7 @@ ggsave( # PLOT TABLES log_info("Saving plot table") vcf %>% - group_by(REGION, synonimous) %>% + group_by(SAMPLE, synonimous) %>% summarise( Freq = sum(ALT_FREQ, na.rm = TRUE) ) %>% @@ -148,9 +149,9 @@ vcf %>% ds = Yes / sum(N_S_position$S) ) %>% ungroup() %>% - left_join(unique(select(vcf, REGION, interval))) %>% + left_join(unique(select(vcf, SAMPLE, interval))) %>% transmute( - sample = REGION, + sample = SAMPLE, DaysSinceFirst = interval, dN = dn, dS = ds, diff --git a/workflow/scripts/report/heatmap.R b/workflow/scripts/report/heatmap.R index 08a2d73..b66cdce 100644 --- a/workflow/scripts/report/heatmap.R +++ b/workflow/scripts/report/heatmap.R @@ -19,7 +19,7 @@ date_order <- read_csv(snakemake@input[["metadata"]]) %>% # Create SNP variable and select useful variables from vcf vcf <- vcf %>% - dplyr::select(variant, REGION, ALT_FREQ) + dplyr::select(variant, SAMPLE, ALT_FREQ) vcf <- vcf %>% pivot_wider( @@ -28,8 +28,8 @@ vcf <- vcf %>% values_fill = 0, values_fn = sum ) %>% - arrange(factor(REGION, levels = date_order)) %>% - column_to_rownames(var = "REGION") + arrange(factor(SAMPLE, levels = date_order)) %>% + column_to_rownames(var = "SAMPLE") log_info("Saving table to create heatmap") write.csv(vcf, snakemake@output[["table"]]) diff --git a/workflow/scripts/report/snp_plots.R b/workflow/scripts/report/snp_plots.R index 44e2ebb..b9df99a 100644 --- a/workflow/scripts/report/snp_plots.R +++ b/workflow/scripts/report/snp_plots.R @@ -27,7 +27,7 @@ metadata <- read_csv(snakemake@input[["metadata"]]) vcf <- read_delim(snakemake@input[["vcf"]]) data <- metadata %>% filter( - ID %in% vcf$REGION + ID %in% vcf$SAMPLE ) %>% dplyr::select( ID, @@ -44,6 +44,7 @@ date_order <- metadata %>% vcf <- vcf %>% dplyr::select( variant, + SAMPLE, REGION, ALT_FREQ, POS @@ -51,10 +52,10 @@ vcf <- vcf %>% # Fill positions without alt frequency with 0 vcf <- vcf %>% - complete(nesting(variant, POS), REGION, fill = list(ALT_FREQ = 0)) + complete(nesting(variant, POS), SAMPLE, fill = list(ALT_FREQ = 0)) # Join variants file and metadata file -vcf <- left_join(vcf, data, by = c("REGION" = "ID")) +vcf <- left_join(vcf, data, by = c("SAMPLE" = "ID")) # Calculate days since first sample vcf <- arrange( @@ -126,7 +127,7 @@ volcano <- cor.df.fill %>% y = trans.p ) + geom_point() + - geom_label_repel(aes(label = label)) + + geom_label_repel(aes(label = label), max.overlaps = 1000) + xlim(c(-1, 1)) + geom_hline( aes(yintercept = -log10(0.05)), @@ -235,7 +236,7 @@ log_info("Saving SNPs trends table") vcf %>% filter(variant %in% subset) %>% transmute( - sample = REGION, + sample = SAMPLE, POS = POS, NV = variant, ALT_FREQ = ALT_FREQ, diff --git a/workflow/scripts/vcf_to_tsv.R b/workflow/scripts/vcf_to_tsv.R index 990f488..f735bab 100644 --- a/workflow/scripts/vcf_to_tsv.R +++ b/workflow/scripts/vcf_to_tsv.R @@ -14,14 +14,14 @@ log_info("Reading data") vcf <- read_tsv(snakemake@input[["ann_vcf"]], comment = "##") tsv <- read_tsv(snakemake@input[["pre_tsv"]]) -tsv["variant"] <- str_extract(vcf$INFO, "p\\.([^|]*)", group = 1) +tsv["variant"] <- str_extract(vcf$INFO, "p\\.([^|]*)", group = 1) # TODO: si hay más de una, se pierde tsv["nuc_variant"] <- str_extract(vcf$INFO, "c\\.([^|]*)", group = 1) tsv <- tsv %>% mutate( variant = case_when( is.na(variant) ~ paste(POS, REF, ">", ALT, sep = ""), - TRUE ~ paste(GFF_FEATURE, ":", variant, sep = "") + TRUE ~ paste(GB_FEATURE, ":", variant, sep = "") ) ) diff --git a/workflow/scripts/weighted_distances.py b/workflow/scripts/weighted_distances.py index 2a5c0e7..dbd8598 100644 --- a/workflow/scripts/weighted_distances.py +++ b/workflow/scripts/weighted_distances.py @@ -46,14 +46,14 @@ def build_ancestor_variant_table(ancestor: Seq, reference: Seq, reference_name: alt.append(reference[i-1]) df = pd.DataFrame({"POS": pos, "ALT": alt}) df["ALT_FREQ"] = 1 # As a reference genome, we assume all positions are monomorphic - df["REGION"] = reference_name + df["SAMPLE"] = reference_name return df def get_frequencies_in_position(variant_table: pd.DataFrame, sample_name: str, position: int, reference: Seq) -> Tuple[float]: frequencies = {alt: 0. for alt in variant_table["ALT"].unique()} alt_keys = sorted(frequencies.keys()) - sample_variant_table = variant_table[variant_table["REGION"] == sample_name] + sample_variant_table = variant_table[variant_table["SAMPLE"] == sample_name] # If the position has polimorphisims, allele frequencies are captured if position in sample_variant_table["POS"].values: alleles = sample_variant_table[sample_variant_table["POS"] == position] From cd4d0e40af9e007a95a42850370cacb54ee6f4d5 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Fri, 5 Sep 2025 14:28:53 +0200 Subject: [PATCH 034/138] Fix iVar output file path --- workflow/rules/vaf.smk | 3 ++- 1 file changed, 2 insertions(+), 1 deletion(-) diff --git a/workflow/rules/vaf.smk b/workflow/rules/vaf.smk index b06c76c..2ed084b 100644 --- a/workflow/rules/vaf.smk +++ b/workflow/rules/vaf.smk @@ -42,12 +42,13 @@ rule snps_to_ancestor: -f renamed_reference.fasta \ {input.bam} \ | ivar variants \ - -p {wildcards.sample} \ + -p variants \ -q {params.ivar_quality} \ -t {params.ivar_freq} \ -m {params.ivar_depth} \ -g {input.gff} \ -r renamed_reference.fasta + mv variants.tsv {output.tsv:q} """ From 3a6fc5623adfc965b51760d67d34594878219444 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Fri, 5 Sep 2025 14:34:05 +0200 Subject: [PATCH 035/138] Add conda directive --- workflow/rules/vaf.smk | 1 + 1 file changed, 1 insertion(+) diff --git a/workflow/rules/vaf.smk b/workflow/rules/vaf.smk index 2ed084b..f452393 100644 --- a/workflow/rules/vaf.smk +++ b/workflow/rules/vaf.smk @@ -197,6 +197,7 @@ rule vcf_to_tsv: rule compile_variants: threads: 1 + conda: "../envs/renv.yaml" input: expand(OUTDIR/"{sample}.masked.filtered.tsv", sample=iter_samples()) output: tsv = OUTDIR/f"{OUTPUT_NAME}.masked.filtered.tsv" From d47ffe5abf3b561ac7ca12cce8d707efec8970d4 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Fri, 5 Sep 2025 14:38:59 +0200 Subject: [PATCH 036/138] Fix sample name detection --- workflow/scripts/compile_variants.R | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/workflow/scripts/compile_variants.R b/workflow/scripts/compile_variants.R index d85654e..b8c442f 100644 --- a/workflow/scripts/compile_variants.R +++ b/workflow/scripts/compile_variants.R @@ -14,7 +14,7 @@ tables <- lapply( snakemake@input, function(path) { read_tsv(path) %>% - mutate(SAMPLE = sub('\\.tsv$', '', path)) + mutate(SAMPLE = sub('\\.masked.filtered.tsv$', '', basename(path))) } ) From 970836db74ca6b751826db2ebb0136292d282298 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Mon, 8 Sep 2025 13:10:14 +0200 Subject: [PATCH 037/138] Nest vaf intermediate files --- workflow/rules/vaf.smk | 34 +++++++++++++++++----------------- 1 file changed, 17 insertions(+), 17 deletions(-) diff --git a/workflow/rules/vaf.smk b/workflow/rules/vaf.smk index f452393..f588736 100644 --- a/workflow/rules/vaf.smk +++ b/workflow/rules/vaf.smk @@ -14,7 +14,7 @@ rule snps_to_ancestor: bam = get_input_bam, gff = OUTDIR/"reference.gff3" output: - tsv = temp(OUTDIR/"{sample}.tsv") + tsv = temp(OUTDIR/"vaf"/"{sample}.tsv") log: LOGDIR / "snps_to_ancestor" / "{sample}.log.txt" shell: @@ -74,10 +74,10 @@ rule mask_tsv: params: mask_class = ["mask"] input: - tsv = OUTDIR/"{sample}.tsv", + tsv = OUTDIR/"vaf"/"{sample}.tsv", vcf = lambda wildcards: select_problematic_vcf() output: - masked_tsv = temp(OUTDIR/"{sample}.masked.tsv") + masked_tsv = temp(OUTDIR/"vaf"/"{sample}.masked.tsv") log: LOGDIR / "mask_tsv" / "{sample}.log.txt" script: @@ -92,10 +92,10 @@ rule filter_tsv: min_alt_rv = 2, min_alt_dp = 2, input: - tsv = OUTDIR/"{sample}.masked.tsv", + tsv = OUTDIR/"vaf"/"{sample}.masked.tsv", annotation = OUTDIR/"annotation.csv" output: - filtered_tsv = temp(OUTDIR/"{sample}.masked.prefiltered.tsv") + filtered_tsv = temp(OUTDIR/"vaf"/"{sample}.masked.prefiltered.tsv") log: LOGDIR / "filter_tsv" / "{sample}.log.txt" script: @@ -108,9 +108,9 @@ rule tsv_to_vcf: params: ref_name = config["ALIGNMENT_REFERENCE"], input: - tsv = OUTDIR/"{sample}.masked.prefiltered.tsv", + tsv = OUTDIR/"vaf"/"{sample}.masked.prefiltered.tsv", output: - vcf = temp(OUTDIR/"{sample}.vcf") + vcf = temp(OUTDIR/"vaf"/"{sample}.vcf") log: LOGDIR / "tsv_to_vcf" / "{sample}.log.txt" script: @@ -125,9 +125,9 @@ rule variants_effect: ref_name = config["ALIGNMENT_REFERENCE"], snpeff_data_dir = (BASE_PATH / "config" / "snpeff").resolve() input: - vcf = OUTDIR/"{sample}.vcf" + vcf = OUTDIR/"vaf"/"{sample}.vcf" output: - ann_vcf = OUTDIR/"{sample}.annotated.vcf" + ann_vcf = OUTDIR/"vaf"/"{sample}.annotated.vcf" log: LOGDIR / "variants_effect" / "{sample}.log.txt" retries: 2 @@ -158,9 +158,9 @@ rule extract_vcf_fields: ], sep = "," input: - vcf = OUTDIR/"{sample}.annotated.vcf" + vcf = OUTDIR/"vaf"/"{sample}.annotated.vcf" output: - tsv = OUTDIR/"{sample}.vcf_fields.tsv" + tsv = OUTDIR/"vaf"/"{sample}.vcf_fields.tsv" log: LOGDIR / "tsv_to_vcf" / "{sample}.log.txt" shell: @@ -172,9 +172,9 @@ rule format_vcf_fields_longer: params: sep = "," input: - tsv = OUTDIR/"{sample}.vcf_fields.tsv" + tsv = OUTDIR/"vaf"/"{sample}.vcf_fields.tsv" output: - tsv = OUTDIR/"{sample}.vcf_fields.longer.tsv" + tsv = OUTDIR/"vaf"/"{sample}.vcf_fields.longer.tsv" log: LOGDIR / "format_vcf_fields_longer" / "{sample}.log.txt" script: @@ -185,10 +185,10 @@ rule vcf_to_tsv: threads: 1 conda: "../envs/renv.yaml" input: - ann_vcf = OUTDIR/"{sample}.annotated.vcf", - pre_tsv = OUTDIR/"{sample}.masked.prefiltered.tsv" + ann_vcf = OUTDIR/"vaf"/"{sample}.annotated.vcf", + pre_tsv = OUTDIR/"vaf"/"{sample}.masked.prefiltered.tsv" output: - tsv = OUTDIR/"{sample}.masked.filtered.tsv" + tsv = OUTDIR/"vaf"/"{sample}.masked.filtered.tsv" log: LOGDIR / "vcf_to_tsv" / "{sample}.log.txt" script: @@ -198,7 +198,7 @@ rule vcf_to_tsv: rule compile_variants: threads: 1 conda: "../envs/renv.yaml" - input: expand(OUTDIR/"{sample}.masked.filtered.tsv", sample=iter_samples()) + input: expand(OUTDIR/"vaf"/"{sample}.masked.filtered.tsv", sample=iter_samples()) output: tsv = OUTDIR/f"{OUTPUT_NAME}.masked.filtered.tsv" log: From 5bfdc5e348f8c4bcf24b6b163355cc72441048a1 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Mon, 8 Sep 2025 13:13:21 +0200 Subject: [PATCH 038/138] Add rule to compile extracted VCF fields --- workflow/rules/vaf.smk | 13 +++++++++++ workflow/scripts/compile_vcf_fields_longer.R | 23 ++++++++++++++++++++ 2 files changed, 36 insertions(+) create mode 100644 workflow/scripts/compile_vcf_fields_longer.R diff --git a/workflow/rules/vaf.smk b/workflow/rules/vaf.smk index f588736..76f2f8e 100644 --- a/workflow/rules/vaf.smk +++ b/workflow/rules/vaf.smk @@ -181,6 +181,19 @@ rule format_vcf_fields_longer: "../scripts/format_vcf_fields_longer.R" +rule compile_vcf_fields_longer: + threads: 1 + conda: "../envs/renv.yaml" + input: + tsv = OUTDIR/"vaf"/"{sample}.vcf_fields.longer.tsv" + output: + tsv = OUTDIR/f"{OUTPUT_NAME}.vcf_fields.longer.tsv" + log: + LOGDIR / "compile_vcf_fields_longer" / "log.txt" + script: + "../scripts/compile_vcf_fields_longer.R" + + rule vcf_to_tsv: threads: 1 conda: "../envs/renv.yaml" diff --git a/workflow/scripts/compile_vcf_fields_longer.R b/workflow/scripts/compile_vcf_fields_longer.R new file mode 100644 index 0000000..73dd243 --- /dev/null +++ b/workflow/scripts/compile_vcf_fields_longer.R @@ -0,0 +1,23 @@ +#!/usr/bin/env Rscript + +# Write stdout and stderr to log file +log <- file(snakemake@log[[1]], open = "wt") +sink(log, type = "message") +sink(log, type = "output") + +library(tidyverse) +library(logger) +log_threshold(INFO) + +log_info("Reading input partial tables with VCF extracted fields and adding sample column") +tables <- lapply( + snakemake@input, + function(path) { + read_tsv(path) %>% + mutate(SAMPLE = sub('\\.vcf_fields.longer.tsv$', '', basename(path))) + } +) + +log_info("Binding and writing table") +bind_rows(tables) %>% + write_tsv(snakemake@output$tsv) From d3b7232cbe1db0eb835a7a21049d9ae5ef2ba5b6 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Mon, 8 Sep 2025 13:15:26 +0200 Subject: [PATCH 039/138] Fix input path typo --- workflow/rules/vaf.smk | 3 +-- 1 file changed, 1 insertion(+), 2 deletions(-) diff --git a/workflow/rules/vaf.smk b/workflow/rules/vaf.smk index 76f2f8e..fcb2dc7 100644 --- a/workflow/rules/vaf.smk +++ b/workflow/rules/vaf.smk @@ -184,8 +184,7 @@ rule format_vcf_fields_longer: rule compile_vcf_fields_longer: threads: 1 conda: "../envs/renv.yaml" - input: - tsv = OUTDIR/"vaf"/"{sample}.vcf_fields.longer.tsv" + input: expand(OUTDIR/"vaf"/"{sample}.vcf_fields.longer.tsv", sample=iter_samples()) output: tsv = OUTDIR/f"{OUTPUT_NAME}.vcf_fields.longer.tsv" log: From 93dda40f2ea8d40b547396b1f35f00ac68a27663 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Mon, 8 Sep 2025 14:48:22 +0200 Subject: [PATCH 040/138] Add VCF field This provides an additional column to avoid rows being duplicate due to different annotation --- workflow/rules/vaf.smk | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/workflow/rules/vaf.smk b/workflow/rules/vaf.smk index fcb2dc7..e3655a5 100644 --- a/workflow/rules/vaf.smk +++ b/workflow/rules/vaf.smk @@ -154,7 +154,7 @@ rule extract_vcf_fields: extract_columns = [ "CHROM", "POS", "REF", "ALT", '"ANN[*].EFFECT"', '"ANN[*].IMPACT"', '"ANN[*].BIOTYPE"', - '"ANN[*].GENE"', '"ANN[*].GENEID"', '"ANN[*].FEATURE"', '"ANN[*].HGVS_P"', '"ANN[*].HGVS_C"' + '"ANN[*].GENE"', '"ANN[*].GENEID"', '"ANN[*].FEATURE"', '"ANN[*].FEATUREID"', '"ANN[*].HGVS_P"', '"ANN[*].HGVS_C"' ], sep = "," input: From 4bf29a6ea70342b4e45e4fc3c8b343eb36881f68 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Mon, 8 Sep 2025 16:31:14 +0200 Subject: [PATCH 041/138] Format extracted VCF fields from the SnpEff annotation --- config/config.yaml | 20 ++++++++++++++++++++ workflow/rules/vaf.smk | 13 ++++++------- workflow/scripts/format_vcf_fields_longer.R | 15 +++++++++++++++ 3 files changed, 41 insertions(+), 7 deletions(-) diff --git a/config/config.yaml b/config/config.yaml index 63f56e3..3973f3e 100644 --- a/config/config.yaml +++ b/config/config.yaml @@ -20,6 +20,26 @@ VC: IVAR_QUALITY: 20 IVAR_FREQ: 0.05 IVAR_DEPTH: 30 +ANNOTATION: + SNPEFF_COLS: + CHROM: CHROM + POS: POS + REF: REF + ALT: ALT + EFFECT: "ANN[*].EFFECT" + IMPACT: "ANN[*].IMPACT" + BIOTYPE: "ANN[*].BIOTYPE" + GENE: "ANN[*].GENE" + GENEID: "ANN[*].GENEID" + FEATURE: "ANN[*].FEATURE" + FEATUREID: "ANN[*].FEATUREID" + HGVS_P: "ANN[*].HGVS_P" + HGVS_C: "ANN[*].HGVS_C" + FILTER_INCLUDE: # see: https://pcingola.github.io/SnpEff/adds/VCFannotationformat_v1.0.pdf + # IMPACT: [HIGH, MODERATE, LOW] + FILTER_EXCLUDE: + # EFFECT: [upstream_gene_variant, downstream_gene_variant] + VARIANT_NAME_PATTERN: "{GENE}:{coalesce(HGVS_P, HGVS_C)}" # coalesce finds the first non-missing element DEMIX: PATHOGEN: "SARS-CoV-2" MIN_QUALITY: 20 diff --git a/workflow/rules/vaf.smk b/workflow/rules/vaf.smk index e3655a5..e678c0e 100644 --- a/workflow/rules/vaf.smk +++ b/workflow/rules/vaf.smk @@ -151,12 +151,8 @@ rule extract_vcf_fields: threads: 1 conda: "../envs/snpeff.yaml" params: - extract_columns = [ - "CHROM", "POS", "REF", "ALT", - '"ANN[*].EFFECT"', '"ANN[*].IMPACT"', '"ANN[*].BIOTYPE"', - '"ANN[*].GENE"', '"ANN[*].GENEID"', '"ANN[*].FEATURE"', '"ANN[*].FEATUREID"', '"ANN[*].HGVS_P"', '"ANN[*].HGVS_C"' - ], - sep = "," + extract_columns = [f"'{col}'" for col in config["ANNOTATION"]["SNPEFF_COLS"].values()], + sep = ",", input: vcf = OUTDIR/"vaf"/"{sample}.annotated.vcf" output: @@ -170,7 +166,10 @@ rule extract_vcf_fields: rule format_vcf_fields_longer: conda: "../envs/renv.yaml" params: - sep = "," + colnames_mapping = config["ANNOTATION"]["SNPEFF_COLS"], + filter_include = config["ANNOTATION"]["FILTER_INCLUDE"], + variant_name_pattern = config["ANNOTATION"]["VARIANT_NAME_PATTERN"], + sep = ",", input: tsv = OUTDIR/"vaf"/"{sample}.vcf_fields.tsv" output: diff --git a/workflow/scripts/format_vcf_fields_longer.R b/workflow/scripts/format_vcf_fields_longer.R index fbe314d..1ac757d 100644 --- a/workflow/scripts/format_vcf_fields_longer.R +++ b/workflow/scripts/format_vcf_fields_longer.R @@ -9,4 +9,19 @@ library(tidyverse) read_tsv(snakemake@input$tsv) %>% separate_rows(contains("[*]"), sep = snakemake@params$sep) %>% + rename(all_of(unlist(snakemake@params$colnames_mapping))) %>% + filter( + !!!map2( + names(snakemake@params$filter_include), + snakemake@params$filter_include, + ~ expr(.data[[!!.x]] %in% !!.y) + ), + !!!map2( + names(snakemake@params$filter_exclude), + snakemake@params$filter_exclude, + ~ expr(!(.data[[!!.x]] %in% !!.y)) + ) + ) %>% + distinct() %>% + mutate(VARIANT_NAME = str_glue(snakemake@params$variant_name_pattern)) %>% write_tsv(snakemake@output$tsv) From e8d56cec6ce95906c3b8f56693dc3ae4ca159ad4 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Mon, 8 Sep 2025 16:33:34 +0200 Subject: [PATCH 042/138] Avoid wildcard expansion --- workflow/rules/vaf.smk | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/workflow/rules/vaf.smk b/workflow/rules/vaf.smk index e678c0e..4fbd4aa 100644 --- a/workflow/rules/vaf.smk +++ b/workflow/rules/vaf.smk @@ -168,7 +168,7 @@ rule format_vcf_fields_longer: params: colnames_mapping = config["ANNOTATION"]["SNPEFF_COLS"], filter_include = config["ANNOTATION"]["FILTER_INCLUDE"], - variant_name_pattern = config["ANNOTATION"]["VARIANT_NAME_PATTERN"], + variant_name_pattern = lambda wildcards: config["ANNOTATION"]["VARIANT_NAME_PATTERN"], # lambda to deactivate automatic wildcard expansion in pattern sep = ",", input: tsv = OUTDIR/"vaf"/"{sample}.vcf_fields.tsv" From 77c33fa482c589fb41cc86dfb4d3d4e3866810a8 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Mon, 8 Sep 2025 16:57:08 +0200 Subject: [PATCH 043/138] Add missing filter param --- workflow/rules/vaf.smk | 1 + 1 file changed, 1 insertion(+) diff --git a/workflow/rules/vaf.smk b/workflow/rules/vaf.smk index 4fbd4aa..3e94b1c 100644 --- a/workflow/rules/vaf.smk +++ b/workflow/rules/vaf.smk @@ -168,6 +168,7 @@ rule format_vcf_fields_longer: params: colnames_mapping = config["ANNOTATION"]["SNPEFF_COLS"], filter_include = config["ANNOTATION"]["FILTER_INCLUDE"], + filter_exclude = config["ANNOTATION"]["FILTER_EXCLUDE"], variant_name_pattern = lambda wildcards: config["ANNOTATION"]["VARIANT_NAME_PATTERN"], # lambda to deactivate automatic wildcard expansion in pattern sep = ",", input: From ab826800886c55f0ec7f489e8d4a4d9b984438e8 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Mon, 8 Sep 2025 17:06:36 +0200 Subject: [PATCH 044/138] Explicitly assign missing values --- workflow/scripts/format_vcf_fields_longer.R | 3 ++- 1 file changed, 2 insertions(+), 1 deletion(-) diff --git a/workflow/scripts/format_vcf_fields_longer.R b/workflow/scripts/format_vcf_fields_longer.R index 1ac757d..5b25031 100644 --- a/workflow/scripts/format_vcf_fields_longer.R +++ b/workflow/scripts/format_vcf_fields_longer.R @@ -8,7 +8,8 @@ sink(log, type = "output") library(tidyverse) read_tsv(snakemake@input$tsv) %>% - separate_rows(contains("[*]"), sep = snakemake@params$sep) %>% + separate_longer_delim(contains("[*]"), delim = snakemake@params$sep) %>% + mutate(across(contains("[*]"), ~ na_if(., ""))) %>% rename(all_of(unlist(snakemake@params$colnames_mapping))) %>% filter( !!!map2( From ccdc0681acb5d77df3330e3faef79fc7b1b617c1 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Mon, 8 Sep 2025 17:33:39 +0200 Subject: [PATCH 045/138] Get variant name for plots from SnpEff annotation --- workflow/rules/distances.smk | 2 +- workflow/rules/report.smk | 10 ++++---- workflow/rules/vaf.smk | 16 ++++++------ workflow/scripts/compile_variants.R | 2 +- workflow/scripts/merge_annotation.R | 30 ++++++++++++++++++++++ workflow/scripts/report/NV_description.R | 12 ++++----- workflow/scripts/report/evo_plots.R | 2 +- workflow/scripts/report/heatmap.R | 4 +-- workflow/scripts/report/snp_plots.R | 20 +++++++-------- workflow/scripts/vcf_to_tsv.R | 32 ------------------------ 10 files changed, 64 insertions(+), 66 deletions(-) create mode 100644 workflow/scripts/merge_annotation.R delete mode 100644 workflow/scripts/vcf_to_tsv.R diff --git a/workflow/rules/distances.smk b/workflow/rules/distances.smk index 886d6a4..d569bd2 100644 --- a/workflow/rules/distances.smk +++ b/workflow/rules/distances.smk @@ -5,7 +5,7 @@ rule weighted_distances: samples = expand("{sample}", sample = iter_samples()), mask_class = ["mask"] input: - tsv = OUTDIR/f"{OUTPUT_NAME}.masked.filtered.tsv", + tsv = OUTDIR/f"{OUTPUT_NAME}.variants.tsv", vcf = lambda wildcards: select_problematic_vcf(), ancestor = OUTDIR/f"{OUTPUT_NAME}.ancestor.fasta", reference = OUTDIR/"reference.fasta" diff --git a/workflow/rules/report.smk b/workflow/rules/report.smk index 2290f3a..707ceab 100644 --- a/workflow/rules/report.smk +++ b/workflow/rules/report.smk @@ -30,7 +30,7 @@ rule demix_plot: rule heatmap_plot_data: conda: "../envs/renv.yaml" input: - vcf = OUTDIR/f"{OUTPUT_NAME}.masked.filtered.tsv", + vcf = OUTDIR/f"{OUTPUT_NAME}.variants.tsv", metadata = config["METADATA"] output: table = report(REPORT_DIR_TABLES/"heatmap.csv") @@ -46,7 +46,7 @@ rule window: window = config["WINDOW"]["WIDTH"], step = config["WINDOW"]["STEP"] input: - vcf = OUTDIR/f"{OUTPUT_NAME}.masked.filtered.tsv", + vcf = OUTDIR/f"{OUTPUT_NAME}.variants.tsv", gb = OUTDIR/"reference.gb", features = config["FEATURES_JSON"] output: @@ -105,7 +105,7 @@ rule general_NV_description: coordinates = config["COORDINATES_JSON"], regions = config["PLOT_GENOME_REGIONS"], window = OUTDIR/f"{OUTPUT_NAME}.window.csv", - vcf = OUTDIR/f"{OUTPUT_NAME}.masked.filtered.tsv", + vcf = OUTDIR/f"{OUTPUT_NAME}.variants.tsv", metadata = config["METADATA"] output: fig = report(REPORT_DIR_PLOTS/"figure_5a.png"), @@ -230,7 +230,7 @@ rule evo_plots: design = config["PLOTS"] input: N_S = OUTDIR/f"{OUTPUT_NAME}.ancestor.N_S.sites.csv", - vcf = OUTDIR/f"{OUTPUT_NAME}.masked.filtered.tsv", + vcf = OUTDIR/f"{OUTPUT_NAME}.variants.tsv", metadata = config["METADATA"] output: plot = report(REPORT_DIR_PLOTS/"figure_11.png"), @@ -249,7 +249,7 @@ rule snp_plots: cor_method = config["COR"]["METHOD"], cor_exact = config["COR"]["EXACT"] input: - vcf = OUTDIR/f"{OUTPUT_NAME}.masked.filtered.tsv", + vcf = OUTDIR/f"{OUTPUT_NAME}.variants.tsv", metadata = config["METADATA"] output: pseudovolcano = report(REPORT_DIR_PLOTS/"figure_6.png"), diff --git a/workflow/rules/vaf.smk b/workflow/rules/vaf.smk index 3e94b1c..dde81c4 100644 --- a/workflow/rules/vaf.smk +++ b/workflow/rules/vaf.smk @@ -193,26 +193,26 @@ rule compile_vcf_fields_longer: "../scripts/compile_vcf_fields_longer.R" -rule vcf_to_tsv: +rule merge_annotation: threads: 1 conda: "../envs/renv.yaml" input: - ann_vcf = OUTDIR/"vaf"/"{sample}.annotated.vcf", - pre_tsv = OUTDIR/"vaf"/"{sample}.masked.prefiltered.tsv" + tsv = OUTDIR/"vaf"/"{sample}.masked.tsv", + annot = OUTDIR/"vaf"/"{sample}.vcf_fields.longer.tsv", output: - tsv = OUTDIR/"vaf"/"{sample}.masked.filtered.tsv" + tsv = OUTDIR/"vaf"/"{sample}.variants.tsv" log: - LOGDIR / "vcf_to_tsv" / "{sample}.log.txt" + LOGDIR / "merge_annotation" / "{sample}.log.txt" script: - "../scripts/vcf_to_tsv.R" + "../scripts/merge_annotation.R" rule compile_variants: threads: 1 conda: "../envs/renv.yaml" - input: expand(OUTDIR/"vaf"/"{sample}.masked.filtered.tsv", sample=iter_samples()) + input: expand(OUTDIR/"vaf"/"{sample}.variants.tsv", sample=iter_samples()) output: - tsv = OUTDIR/f"{OUTPUT_NAME}.masked.filtered.tsv" + tsv = OUTDIR/f"{OUTPUT_NAME}.variants.tsv" log: LOGDIR / "compile_variants" / "log.txt" script: diff --git a/workflow/scripts/compile_variants.R b/workflow/scripts/compile_variants.R index b8c442f..7e7baaa 100644 --- a/workflow/scripts/compile_variants.R +++ b/workflow/scripts/compile_variants.R @@ -14,7 +14,7 @@ tables <- lapply( snakemake@input, function(path) { read_tsv(path) %>% - mutate(SAMPLE = sub('\\.masked.filtered.tsv$', '', basename(path))) + mutate(SAMPLE = sub('\\.variants.tsv$', '', basename(path))) } ) diff --git a/workflow/scripts/merge_annotation.R b/workflow/scripts/merge_annotation.R new file mode 100644 index 0000000..3483e20 --- /dev/null +++ b/workflow/scripts/merge_annotation.R @@ -0,0 +1,30 @@ +#!/usr/bin/env Rscript + +# Write stdout and stderr to log file +log <- file(snakemake@log[[1]], open = "wt") +sink(log, type = "message") +sink(log, type = "output") + +library(tidyverse) +library(logger) +log_threshold(INFO) + +log_info("Reading tables") +variants <- read_tsv(snakemake@input$tsv) +annotation <- read_tsv( + snakemake@input$annot, + col_select = c("CHROM", "POS", "REF", "ALT", "VARIANT_NAME") +) + +log_info("Merging tables") +merged <- left_join( + variants, + annotation, + by = c("CHROM", "POS", "REF", "ALT") +) + +log_info("Saving results") +write_tsv( + tsv, + snakemake@output$tsv +) diff --git a/workflow/scripts/report/NV_description.R b/workflow/scripts/report/NV_description.R index 5c4b398..047a512 100644 --- a/workflow/scripts/report/NV_description.R +++ b/workflow/scripts/report/NV_description.R @@ -40,7 +40,7 @@ date_order <- metadata %>% empty_vcf <- tibble( SAMPLE = date_order, REGION = as.character(NA), - variant = as.character(NA), + VARIANT_NAME = as.character(NA), ALT_FREQ = as.numeric(NA), GB_FEATURE = as.character(NA), synonimous = as.character(NA), @@ -55,7 +55,7 @@ vcf <- vcf %>% dplyr::select( SAMPLE, REGION, - variant, + VARIANT_NAME, ALT_FREQ, GB_FEATURE, synonimous, @@ -414,14 +414,14 @@ vcf %>% select( SAMPLE, POS, - variant, + VARIANT_NAME, ALT_FREQ, NV_class, group ) %>% rename( sample = SAMPLE, - Variant = variant, + Variant = VARIANT_NAME, Class = group ) %>% filter(ALT_FREQ > 0) %>% @@ -464,11 +464,11 @@ vcf_snp %>% # Stats for reporting n_indels <- vcf %>% filter(NV_class == "INDEL") %>% - pull(variant) %>% + pull(VARIANT_NAME) %>% unique() %>% length() -n_snv <- length(unique(vcf$variant)) - n_indels +n_snv <- length(unique(vcf$VARIANT_NAME)) - n_indels model <- lm(n ~ CollectionDate, data = figur_SNP_table) # Calculate correlation, if possible diff --git a/workflow/scripts/report/evo_plots.R b/workflow/scripts/report/evo_plots.R index 586328e..9e1e1db 100644 --- a/workflow/scripts/report/evo_plots.R +++ b/workflow/scripts/report/evo_plots.R @@ -22,7 +22,7 @@ N_S_position <- read_delim(snakemake@input[["N_S"]]) vcf <- vcf %>% dplyr::select( SAMPLE, - variant, + VARIANT_NAME, REGION, ALT_FREQ, GB_FEATURE, diff --git a/workflow/scripts/report/heatmap.R b/workflow/scripts/report/heatmap.R index b66cdce..bfcdde7 100644 --- a/workflow/scripts/report/heatmap.R +++ b/workflow/scripts/report/heatmap.R @@ -19,11 +19,11 @@ date_order <- read_csv(snakemake@input[["metadata"]]) %>% # Create SNP variable and select useful variables from vcf vcf <- vcf %>% - dplyr::select(variant, SAMPLE, ALT_FREQ) + dplyr::select(VARIANT_NAME, SAMPLE, ALT_FREQ) vcf <- vcf %>% pivot_wider( - names_from = variant, + names_from = VARIANT_NAME, values_from = ALT_FREQ, values_fill = 0, values_fn = sum diff --git a/workflow/scripts/report/snp_plots.R b/workflow/scripts/report/snp_plots.R index b9df99a..b7f128b 100644 --- a/workflow/scripts/report/snp_plots.R +++ b/workflow/scripts/report/snp_plots.R @@ -43,7 +43,7 @@ date_order <- metadata %>% # Simplify features names and create SNP variable vcf <- vcf %>% dplyr::select( - variant, + VARIANT_NAME, SAMPLE, REGION, ALT_FREQ, @@ -52,7 +52,7 @@ vcf <- vcf %>% # Fill positions without alt frequency with 0 vcf <- vcf %>% - complete(nesting(variant, POS), SAMPLE, fill = list(ALT_FREQ = 0)) + complete(nesting(VARIANT_NAME, POS), SAMPLE, fill = list(ALT_FREQ = 0)) # Join variants file and metadata file vcf <- left_join(vcf, data, by = c("SAMPLE" = "ID")) @@ -72,7 +72,7 @@ vcf <- arrange( # Get list with all different polymorphisms SNPs <- pull( vcf, - variant + VARIANT_NAME ) %>% unique() @@ -89,7 +89,7 @@ cor.df.fill <- lapply( function(snp) { df <- filter( vcf, - variant == snp + VARIANT_NAME == snp ) test <- cor.test( @@ -161,14 +161,14 @@ sign <- filter( # SNPs which are in positions with more than one alternative allele dup <- vcf %>% select( - variant, + VARIANT_NAME, POS ) %>% unique() %>% group_by(POS) %>% filter(n() > 1) %>% ungroup() %>% - pull(variant) %>% + pull(VARIANT_NAME) %>% unique() subset <- c(sign, dup) %>% @@ -179,12 +179,12 @@ plot.height <- ceiling(length(subset) / 4) * 42 log_info("Plotting SNPs trends in time") panel <- vcf %>% - filter(variant %in% subset) %>% + filter(VARIANT_NAME %in% subset) %>% ggplot() + aes( x = interval, y = ALT_FREQ, - color = variant + color = VARIANT_NAME ) + scale_color_manual(values = sample(color, length(subset))) + geom_point() + @@ -234,11 +234,11 @@ cor.df.fill %>% log_info("Saving SNPs trends table") vcf %>% - filter(variant %in% subset) %>% + filter(VARIANT_NAME %in% subset) %>% transmute( sample = SAMPLE, POS = POS, - NV = variant, + NV = VARIANT_NAME, ALT_FREQ = ALT_FREQ, DaysSinceFirst = interval ) %>% diff --git a/workflow/scripts/vcf_to_tsv.R b/workflow/scripts/vcf_to_tsv.R deleted file mode 100644 index f735bab..0000000 --- a/workflow/scripts/vcf_to_tsv.R +++ /dev/null @@ -1,32 +0,0 @@ -#!/usr/bin/env Rscript - -# Write stdout and stderr to log file -log <- file(snakemake@log[[1]], open = "wt") -sink(log, type = "message") -sink(log, type = "output") - -library(tidyverse) -library(logger) -log_threshold(INFO) - -# read data -log_info("Reading data") -vcf <- read_tsv(snakemake@input[["ann_vcf"]], comment = "##") -tsv <- read_tsv(snakemake@input[["pre_tsv"]]) - -tsv["variant"] <- str_extract(vcf$INFO, "p\\.([^|]*)", group = 1) # TODO: si hay más de una, se pierde -tsv["nuc_variant"] <- str_extract(vcf$INFO, "c\\.([^|]*)", group = 1) - -tsv <- tsv %>% - mutate( - variant = case_when( - is.na(variant) ~ paste(POS, REF, ">", ALT, sep = ""), - TRUE ~ paste(GB_FEATURE, ":", variant, sep = "") - ) - ) - -log_info("Saving results") -write_tsv( - tsv, - snakemake@output[["tsv"]] -) From 29f25e338b8bc13fd04e52ec4dfcec503387f11c Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Mon, 8 Sep 2025 17:50:47 +0200 Subject: [PATCH 046/138] Fix reference name mismatch --- workflow/rules/vaf.smk | 2 ++ workflow/scripts/merge_annotation.R | 9 ++++++--- 2 files changed, 8 insertions(+), 3 deletions(-) diff --git a/workflow/rules/vaf.smk b/workflow/rules/vaf.smk index dde81c4..640eb72 100644 --- a/workflow/rules/vaf.smk +++ b/workflow/rules/vaf.smk @@ -196,6 +196,8 @@ rule compile_vcf_fields_longer: rule merge_annotation: threads: 1 conda: "../envs/renv.yaml" + params: + ref_name = config["ALIGNMENT_REFERENCE"], input: tsv = OUTDIR/"vaf"/"{sample}.masked.tsv", annot = OUTDIR/"vaf"/"{sample}.vcf_fields.longer.tsv", diff --git a/workflow/scripts/merge_annotation.R b/workflow/scripts/merge_annotation.R index 3483e20..351efaf 100644 --- a/workflow/scripts/merge_annotation.R +++ b/workflow/scripts/merge_annotation.R @@ -9,8 +9,11 @@ library(tidyverse) library(logger) log_threshold(INFO) -log_info("Reading tables") -variants <- read_tsv(snakemake@input$tsv) +log_info("Reading variants table, replacing REGION with the reference name") +variants <- read_tsv(snakemake@input$tsv) %>% + mutate(REGION = snakemake@params$ref_name) + +log_info("Reading annotation table") annotation <- read_tsv( snakemake@input$annot, col_select = c("CHROM", "POS", "REF", "ALT", "VARIANT_NAME") @@ -20,7 +23,7 @@ log_info("Merging tables") merged <- left_join( variants, annotation, - by = c("CHROM", "POS", "REF", "ALT") + by = c("REGION" = "CHROM", "POS", "REF", "ALT") ) log_info("Saving results") From 02533c8b470a71cecf2646f1ff9d37376b60a348 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Mon, 8 Sep 2025 17:53:06 +0200 Subject: [PATCH 047/138] Annotate distinct variants --- workflow/scripts/merge_annotation.R | 5 +++-- 1 file changed, 3 insertions(+), 2 deletions(-) diff --git a/workflow/scripts/merge_annotation.R b/workflow/scripts/merge_annotation.R index 351efaf..c7b68ea 100644 --- a/workflow/scripts/merge_annotation.R +++ b/workflow/scripts/merge_annotation.R @@ -17,7 +17,8 @@ log_info("Reading annotation table") annotation <- read_tsv( snakemake@input$annot, col_select = c("CHROM", "POS", "REF", "ALT", "VARIANT_NAME") -) +) %>% + distinct() log_info("Merging tables") merged <- left_join( @@ -28,6 +29,6 @@ merged <- left_join( log_info("Saving results") write_tsv( - tsv, + merged, snakemake@output$tsv ) From cc6002b152f5e117c6411f30f497d676f735a077 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Mon, 8 Sep 2025 17:58:22 +0200 Subject: [PATCH 048/138] Fix merge annotation target --- workflow/rules/vaf.smk | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/workflow/rules/vaf.smk b/workflow/rules/vaf.smk index 640eb72..d394ae0 100644 --- a/workflow/rules/vaf.smk +++ b/workflow/rules/vaf.smk @@ -199,7 +199,7 @@ rule merge_annotation: params: ref_name = config["ALIGNMENT_REFERENCE"], input: - tsv = OUTDIR/"vaf"/"{sample}.masked.tsv", + tsv = OUTDIR/"vaf"/"{sample}.masked.prefiltered.tsv", annot = OUTDIR/"vaf"/"{sample}.vcf_fields.longer.tsv", output: tsv = OUTDIR/"vaf"/"{sample}.variants.tsv" From b9d393efc6aa42fdfb8fcb9b4276916fbd1af3c4 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Mon, 8 Sep 2025 18:21:00 +0200 Subject: [PATCH 049/138] Collapse different mutation name rows to a single field --- workflow/scripts/merge_annotation.R | 5 ++++- 1 file changed, 4 insertions(+), 1 deletion(-) diff --git a/workflow/scripts/merge_annotation.R b/workflow/scripts/merge_annotation.R index c7b68ea..e6f5803 100644 --- a/workflow/scripts/merge_annotation.R +++ b/workflow/scripts/merge_annotation.R @@ -18,7 +18,10 @@ annotation <- read_tsv( snakemake@input$annot, col_select = c("CHROM", "POS", "REF", "ALT", "VARIANT_NAME") ) %>% - distinct() + distinct() %>% + group_by(CHROM, POS, REF, ALT) %>% + mutate(VARIANT_NAME = paste(VARIANT_NAME, collapse = "|")) %>% + ungroup() log_info("Merging tables") merged <- left_join( From 087e84595987e7ff4cacc870215976f47a915406 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Mon, 8 Sep 2025 18:21:20 +0200 Subject: [PATCH 050/138] Filter out upstream and downstream variants by default --- config/config.yaml | 4 ++-- 1 file changed, 2 insertions(+), 2 deletions(-) diff --git a/config/config.yaml b/config/config.yaml index 3973f3e..9197f74 100644 --- a/config/config.yaml +++ b/config/config.yaml @@ -38,8 +38,8 @@ ANNOTATION: FILTER_INCLUDE: # see: https://pcingola.github.io/SnpEff/adds/VCFannotationformat_v1.0.pdf # IMPACT: [HIGH, MODERATE, LOW] FILTER_EXCLUDE: - # EFFECT: [upstream_gene_variant, downstream_gene_variant] - VARIANT_NAME_PATTERN: "{GENE}:{coalesce(HGVS_P, HGVS_C)}" # coalesce finds the first non-missing element + EFFECT: [upstream_gene_variant, downstream_gene_variant] + VARIANT_NAME_PATTERN: "{GENE}:{coalesce(HGVS_P, HGVS_C)}" # dplyr's coalesce finds the first non-missing element DEMIX: PATHOGEN: "SARS-CoV-2" MIN_QUALITY: 20 From 3c97af8546b44df585c795f7031cf1cdee45feb9 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 9 Sep 2025 11:39:19 +0200 Subject: [PATCH 051/138] Filter out errors in SnpEff Also replaces empty values ("" in config YAML) with NA in R and adds code comments --- config/config.yaml | 2 ++ workflow/scripts/format_vcf_fields_longer.R | 31 ++++++++++++++++++--- 2 files changed, 29 insertions(+), 4 deletions(-) diff --git a/config/config.yaml b/config/config.yaml index 9197f74..3c66286 100644 --- a/config/config.yaml +++ b/config/config.yaml @@ -35,7 +35,9 @@ ANNOTATION: FEATUREID: "ANN[*].FEATUREID" HGVS_P: "ANN[*].HGVS_P" HGVS_C: "ANN[*].HGVS_C" + ERRORS: "ANN[*].ERRORS" FILTER_INCLUDE: # see: https://pcingola.github.io/SnpEff/adds/VCFannotationformat_v1.0.pdf + ERRORS: [""] # IMPACT: [HIGH, MODERATE, LOW] FILTER_EXCLUDE: EFFECT: [upstream_gene_variant, downstream_gene_variant] diff --git a/workflow/scripts/format_vcf_fields_longer.R b/workflow/scripts/format_vcf_fields_longer.R index 5b25031..accd1ea 100644 --- a/workflow/scripts/format_vcf_fields_longer.R +++ b/workflow/scripts/format_vcf_fields_longer.R @@ -7,22 +7,45 @@ sink(log, type = "output") library(tidyverse) +# Replace "" values with NA in R filter list +# Snakemake passes filters like: list(ERRORS = c("")) +empty.to.na <- function(x) { + x[x == ""] <- NA + x +} +filter.include <- lapply(snakemake@params$filter_include, empty.to.na) +filter.exclude <- lapply(snakemake@params$filter_exclude, empty.to.na) + +# Process input table read_tsv(snakemake@input$tsv) %>% + # Separate -delimited "...[*]..." columns (e.g. ANN[*].EFFECT) separate_longer_delim(contains("[*]"), delim = snakemake@params$sep) %>% + # Replace empty ("") fields with NA mutate(across(contains("[*]"), ~ na_if(., ""))) %>% + # Rename "...[*]..." columns using the provided lookup via Snakemake config rename(all_of(unlist(snakemake@params$colnames_mapping))) %>% + # Apply dynamic filters from the Snakemake config: + # map2 pairs column names (.x) with value vectors (.y) and builds boolean expressions. + # Inside the expr call, !! injects a single value into each expression. + # The resulting list of expressions is spliced with !!! so each becomes its + # own condition as if written directly inside the filter call. filter( + # Keep variants that include the required values in each defined field (e.g. empty ERRORS) !!!map2( - names(snakemake@params$filter_include), - snakemake@params$filter_include, + names(filter.include), + filter.include, ~ expr(.data[[!!.x]] %in% !!.y) ), + # Keep variants that exclude the required values in each defined field (e.g. EFFECT != "upstream_gene_variant") !!!map2( - names(snakemake@params$filter_exclude), - snakemake@params$filter_exclude, + names(filter.exclude), + filter.exclude, ~ expr(!(.data[[!!.x]] %in% !!.y)) ) ) %>% + # Keep unique rows distinct() %>% + # Assign variant name using the pattern defined via Snakemake config mutate(VARIANT_NAME = str_glue(snakemake@params$variant_name_pattern)) %>% + # Write output file write_tsv(snakemake@output$tsv) From 2d6af9eea59d9ea6700b1248947812883ff5dd33 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 9 Sep 2025 12:32:44 +0200 Subject: [PATCH 052/138] Fill empty ANN values with NA to ensure a correct extraction --- workflow/rules/vaf.smk | 2 +- workflow/scripts/format_vcf_fields_longer.R | 4 +--- 2 files changed, 2 insertions(+), 4 deletions(-) diff --git a/workflow/rules/vaf.smk b/workflow/rules/vaf.smk index d394ae0..53f8a8c 100644 --- a/workflow/rules/vaf.smk +++ b/workflow/rules/vaf.smk @@ -160,7 +160,7 @@ rule extract_vcf_fields: log: LOGDIR / "tsv_to_vcf" / "{sample}.log.txt" shell: - 'SnpSift extractFields -s {params.sep:q} {input.vcf:q} {params.extract_columns} >{output.tsv:q} 2>{log:q}' + "SnpSift extractFields -e 'NA' -s {params.sep:q} {input.vcf:q} {params.extract_columns} >{output.tsv:q} 2>{log:q}" rule format_vcf_fields_longer: diff --git a/workflow/scripts/format_vcf_fields_longer.R b/workflow/scripts/format_vcf_fields_longer.R index accd1ea..439388b 100644 --- a/workflow/scripts/format_vcf_fields_longer.R +++ b/workflow/scripts/format_vcf_fields_longer.R @@ -19,9 +19,7 @@ filter.exclude <- lapply(snakemake@params$filter_exclude, empty.to.na) # Process input table read_tsv(snakemake@input$tsv) %>% # Separate -delimited "...[*]..." columns (e.g. ANN[*].EFFECT) - separate_longer_delim(contains("[*]"), delim = snakemake@params$sep) %>% - # Replace empty ("") fields with NA - mutate(across(contains("[*]"), ~ na_if(., ""))) %>% + separate_rows(contains("[*]"), sep = snakemake@params$sep, convert = TRUE) %>% # Rename "...[*]..." columns using the provided lookup via Snakemake config rename(all_of(unlist(snakemake@params$colnames_mapping))) %>% # Apply dynamic filters from the Snakemake config: From ac3512aabdb65b22ab6777e7c46f4b7b241b570e Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 9 Sep 2025 12:43:14 +0200 Subject: [PATCH 053/138] Specify column types --- workflow/scripts/merge_annotation.R | 19 +++++++++++++++++-- 1 file changed, 17 insertions(+), 2 deletions(-) diff --git a/workflow/scripts/merge_annotation.R b/workflow/scripts/merge_annotation.R index e6f5803..7eccb56 100644 --- a/workflow/scripts/merge_annotation.R +++ b/workflow/scripts/merge_annotation.R @@ -10,13 +10,28 @@ library(logger) log_threshold(INFO) log_info("Reading variants table, replacing REGION with the reference name") -variants <- read_tsv(snakemake@input$tsv) %>% +variants <- read_tsv( + snakemake@input$tsv, + col_types = list( + CHROM = col_character(), + POS = col_integer(), + REF = col_character(), + ALT = col_character() + ) +) %>% mutate(REGION = snakemake@params$ref_name) log_info("Reading annotation table") annotation <- read_tsv( snakemake@input$annot, - col_select = c("CHROM", "POS", "REF", "ALT", "VARIANT_NAME") + col_select = c("CHROM", "POS", "REF", "ALT", "VARIANT_NAME"), + col_types = list( + CHROM = col_character(), + POS = col_integer(), + REF = col_character(), + ALT = col_character(), + VARIANT_NAME = col_character() + ) ) %>% distinct() %>% group_by(CHROM, POS, REF, ALT) %>% From 2df2dc41469c6e00c3beaadeb499fa1c171737c1 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 9 Sep 2025 12:47:52 +0200 Subject: [PATCH 054/138] Exclude SnpEff errors and warnings instead of including empty error fields This allows INFO messages --- config/config.yaml | 13 +++++++++++-- 1 file changed, 11 insertions(+), 2 deletions(-) diff --git a/config/config.yaml b/config/config.yaml index 3c66286..fd51332 100644 --- a/config/config.yaml +++ b/config/config.yaml @@ -21,6 +21,7 @@ VC: IVAR_FREQ: 0.05 IVAR_DEPTH: 30 ANNOTATION: + # see: https://pcingola.github.io/SnpEff/adds/VCFannotationformat_v1.0.pdf SNPEFF_COLS: CHROM: CHROM POS: POS @@ -36,11 +37,19 @@ ANNOTATION: HGVS_P: "ANN[*].HGVS_P" HGVS_C: "ANN[*].HGVS_C" ERRORS: "ANN[*].ERRORS" - FILTER_INCLUDE: # see: https://pcingola.github.io/SnpEff/adds/VCFannotationformat_v1.0.pdf - ERRORS: [""] + FILTER_INCLUDE: # IMPACT: [HIGH, MODERATE, LOW] FILTER_EXCLUDE: EFFECT: [upstream_gene_variant, downstream_gene_variant] + ERRORS: + - ERROR_CHROMOSOME_NOT_FOUND + - ERROR_OUT_OF_CHROMOSOME_RANGE + - WARNING_REF_DOES_NOT_MATCH_GENOME + - WARNING_SEQUENCE_NOT_AVAILABLE + - WARNING_TRANSCRIPT_INCOMPLETE + - WARNING_TRANSCRIPT_MULTIPLE_STOP_CODONS + - WARNING_TRANSCRIPT_NO_START_CODON + - WARNING_TRANSCRIPT_NO_STOP_CODON VARIANT_NAME_PATTERN: "{GENE}:{coalesce(HGVS_P, HGVS_C)}" # dplyr's coalesce finds the first non-missing element DEMIX: PATHOGEN: "SARS-CoV-2" From 9c5660e321ffaaed0a006ef90f3f70bf0bddb650 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 9 Sep 2025 13:35:24 +0200 Subject: [PATCH 055/138] Split SnpEff error column Many error/warning/info messages can appear on the same variant --- config/config.yaml | 2 +- workflow/scripts/format_vcf_fields_longer.R | 21 ++++++++++++++++++--- 2 files changed, 19 insertions(+), 4 deletions(-) diff --git a/config/config.yaml b/config/config.yaml index fd51332..8fc7441 100644 --- a/config/config.yaml +++ b/config/config.yaml @@ -36,7 +36,7 @@ ANNOTATION: FEATUREID: "ANN[*].FEATUREID" HGVS_P: "ANN[*].HGVS_P" HGVS_C: "ANN[*].HGVS_C" - ERRORS: "ANN[*].ERRORS" + ERRORS: "ANN[*].ERRORS" # hard-coded column FILTER_INCLUDE: # IMPACT: [HIGH, MODERATE, LOW] FILTER_EXCLUDE: diff --git a/workflow/scripts/format_vcf_fields_longer.R b/workflow/scripts/format_vcf_fields_longer.R index 439388b..8878c65 100644 --- a/workflow/scripts/format_vcf_fields_longer.R +++ b/workflow/scripts/format_vcf_fields_longer.R @@ -7,19 +7,34 @@ sink(log, type = "output") library(tidyverse) -# Replace "" values with NA in R filter list -# Snakemake passes filters like: list(ERRORS = c("")) empty.to.na <- function(x) { x[x == ""] <- NA x } + +separate_rows_if_exist <- function(df, col, sep) { + if (col %in% colnames(df)) { + separate_longer_delim(df, all_of(cols), delim = sep) + } else { + df + } +} + +# Replace "" values with NA in R filter list +# Snakemake passes filters like: list(ERRORS = c("")) filter.include <- lapply(snakemake@params$filter_include, empty.to.na) filter.exclude <- lapply(snakemake@params$filter_exclude, empty.to.na) # Process input table read_tsv(snakemake@input$tsv) %>% # Separate -delimited "...[*]..." columns (e.g. ANN[*].EFFECT) - separate_rows(contains("[*]"), sep = snakemake@params$sep, convert = TRUE) %>% + separate_rows( + contains("[*]"), + sep = snakemake@params$sep, + convert = TRUE + ) %>% + # Separate &-delimited error column (more than one error/warning/info message per row is possible) + separate_rows_if_exist("ERRORS", sep = snakemake@params$sep) %>% # Rename "...[*]..." columns using the provided lookup via Snakemake config rename(all_of(unlist(snakemake@params$colnames_mapping))) %>% # Apply dynamic filters from the Snakemake config: From 394055e6020b1017a65fe091929a631dbd02c762 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 9 Sep 2025 13:36:11 +0200 Subject: [PATCH 056/138] Complete with zero alt frequency including region --- workflow/scripts/report/snp_plots.R | 3 ++- 1 file changed, 2 insertions(+), 1 deletion(-) diff --git a/workflow/scripts/report/snp_plots.R b/workflow/scripts/report/snp_plots.R index b7f128b..be2f7ba 100644 --- a/workflow/scripts/report/snp_plots.R +++ b/workflow/scripts/report/snp_plots.R @@ -52,7 +52,7 @@ vcf <- vcf %>% # Fill positions without alt frequency with 0 vcf <- vcf %>% - complete(nesting(VARIANT_NAME, POS), SAMPLE, fill = list(ALT_FREQ = 0)) + complete(nesting(REGION, VARIANT_NAME, POS), SAMPLE, fill = list(ALT_FREQ = 0)) # Join variants file and metadata file vcf <- left_join(vcf, data, by = c("SAMPLE" = "ID")) @@ -87,6 +87,7 @@ cor.df <- data.frame( cor.df.fill <- lapply( SNPs, function(snp) { + message(snp) df <- filter( vcf, VARIANT_NAME == snp From 0014b306aebddd9ba6a27bbf8f3529437dc683a0 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 9 Sep 2025 13:43:03 +0200 Subject: [PATCH 057/138] Fix error separator --- workflow/scripts/format_vcf_fields_longer.R | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/workflow/scripts/format_vcf_fields_longer.R b/workflow/scripts/format_vcf_fields_longer.R index 8878c65..a01e101 100644 --- a/workflow/scripts/format_vcf_fields_longer.R +++ b/workflow/scripts/format_vcf_fields_longer.R @@ -34,7 +34,7 @@ read_tsv(snakemake@input$tsv) %>% convert = TRUE ) %>% # Separate &-delimited error column (more than one error/warning/info message per row is possible) - separate_rows_if_exist("ERRORS", sep = snakemake@params$sep) %>% + separate_rows_if_exist("ERRORS", sep = "&") %>% # Rename "...[*]..." columns using the provided lookup via Snakemake config rename(all_of(unlist(snakemake@params$colnames_mapping))) %>% # Apply dynamic filters from the Snakemake config: From 6539324803e5ad089488cb059df47679a30461ea Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 9 Sep 2025 13:52:56 +0200 Subject: [PATCH 058/138] Hard-code error column split --- workflow/scripts/format_vcf_fields_longer.R | 10 +--------- 1 file changed, 1 insertion(+), 9 deletions(-) diff --git a/workflow/scripts/format_vcf_fields_longer.R b/workflow/scripts/format_vcf_fields_longer.R index a01e101..4f288c5 100644 --- a/workflow/scripts/format_vcf_fields_longer.R +++ b/workflow/scripts/format_vcf_fields_longer.R @@ -12,14 +12,6 @@ empty.to.na <- function(x) { x } -separate_rows_if_exist <- function(df, col, sep) { - if (col %in% colnames(df)) { - separate_longer_delim(df, all_of(cols), delim = sep) - } else { - df - } -} - # Replace "" values with NA in R filter list # Snakemake passes filters like: list(ERRORS = c("")) filter.include <- lapply(snakemake@params$filter_include, empty.to.na) @@ -34,7 +26,7 @@ read_tsv(snakemake@input$tsv) %>% convert = TRUE ) %>% # Separate &-delimited error column (more than one error/warning/info message per row is possible) - separate_rows_if_exist("ERRORS", sep = "&") %>% + separate_rows("ERRORS", sep = "&") %>% # Rename "...[*]..." columns using the provided lookup via Snakemake config rename(all_of(unlist(snakemake@params$colnames_mapping))) %>% # Apply dynamic filters from the Snakemake config: From 8118df826e97ab8dde58e88f65133488098648c5 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 9 Sep 2025 13:55:16 +0200 Subject: [PATCH 059/138] Fix error column name Hard-code using SnpEff name --- workflow/scripts/format_vcf_fields_longer.R | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/workflow/scripts/format_vcf_fields_longer.R b/workflow/scripts/format_vcf_fields_longer.R index 4f288c5..f05b261 100644 --- a/workflow/scripts/format_vcf_fields_longer.R +++ b/workflow/scripts/format_vcf_fields_longer.R @@ -26,7 +26,7 @@ read_tsv(snakemake@input$tsv) %>% convert = TRUE ) %>% # Separate &-delimited error column (more than one error/warning/info message per row is possible) - separate_rows("ERRORS", sep = "&") %>% + separate_rows("ANN[*].ERRORS", sep = "&") %>% # Rename "...[*]..." columns using the provided lookup via Snakemake config rename(all_of(unlist(snakemake@params$colnames_mapping))) %>% # Apply dynamic filters from the Snakemake config: From c443199900e6ccc1a65405ea965f2e56cb36e728 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 9 Sep 2025 15:18:51 +0200 Subject: [PATCH 060/138] Fix column name --- workflow/scripts/merge_annotation.R | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/workflow/scripts/merge_annotation.R b/workflow/scripts/merge_annotation.R index 7eccb56..b8218f2 100644 --- a/workflow/scripts/merge_annotation.R +++ b/workflow/scripts/merge_annotation.R @@ -13,7 +13,7 @@ log_info("Reading variants table, replacing REGION with the reference name") variants <- read_tsv( snakemake@input$tsv, col_types = list( - CHROM = col_character(), + REGION = col_character(), POS = col_integer(), REF = col_character(), ALT = col_character() From d7ed3a019f683a86b5f7b5b5070aa4396046cbf0 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 9 Sep 2025 15:19:53 +0200 Subject: [PATCH 061/138] Fix filter SnpEff error column Uses a list column instead of multiple rows to avoid issues with multiple messages --- workflow/scripts/format_vcf_fields_longer.R | 14 ++++++++++++-- 1 file changed, 12 insertions(+), 2 deletions(-) diff --git a/workflow/scripts/format_vcf_fields_longer.R b/workflow/scripts/format_vcf_fields_longer.R index f05b261..407e9fb 100644 --- a/workflow/scripts/format_vcf_fields_longer.R +++ b/workflow/scripts/format_vcf_fields_longer.R @@ -19,16 +19,23 @@ filter.exclude <- lapply(snakemake@params$filter_exclude, empty.to.na) # Process input table read_tsv(snakemake@input$tsv) %>% + # Separate -delimited "...[*]..." columns (e.g. ANN[*].EFFECT) separate_rows( contains("[*]"), sep = snakemake@params$sep, convert = TRUE ) %>% - # Separate &-delimited error column (more than one error/warning/info message per row is possible) - separate_rows("ANN[*].ERRORS", sep = "&") %>% + # Rename "...[*]..." columns using the provided lookup via Snakemake config rename(all_of(unlist(snakemake@params$colnames_mapping))) %>% + + # Separate &-delimited error column (more than one error/warning/info message per row is possible) + mutate(split_errors = strsplit(ERRORS, "&")) %>% + # Keep rows with none of the excluded ERRORS terms, if any + filter(map_lgl(split_errors, ~ !any(. %in% filter.exclude[["ERRORS"]]))) %>% + select(-split_errors) %>% + # Apply dynamic filters from the Snakemake config: # map2 pairs column names (.x) with value vectors (.y) and builds boolean expressions. # Inside the expr call, !! injects a single value into each expression. @@ -48,9 +55,12 @@ read_tsv(snakemake@input$tsv) %>% ~ expr(!(.data[[!!.x]] %in% !!.y)) ) ) %>% + # Keep unique rows distinct() %>% + # Assign variant name using the pattern defined via Snakemake config mutate(VARIANT_NAME = str_glue(snakemake@params$variant_name_pattern)) %>% + # Write output file write_tsv(snakemake@output$tsv) From 316bbc2ed1890aba614aae3eda4480409b735b78 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 9 Sep 2025 16:11:55 +0200 Subject: [PATCH 062/138] Fix merger iVar TSV with SnpEff annotation using the same variant format Adds columns REF_VCF and ALT_VCF with VCF-like alleles --- workflow/scripts/merge_annotation.R | 23 +++++++++++++++++++++-- 1 file changed, 21 insertions(+), 2 deletions(-) diff --git a/workflow/scripts/merge_annotation.R b/workflow/scripts/merge_annotation.R index b8218f2..0d77f1d 100644 --- a/workflow/scripts/merge_annotation.R +++ b/workflow/scripts/merge_annotation.R @@ -19,7 +19,21 @@ variants <- read_tsv( ALT = col_character() ) ) %>% - mutate(REGION = snakemake@params$ref_name) + mutate( + REGION = snakemake@params$ref_name, + is_insertion = startsWith(ALT, "+"), + is_deletion = startsWith(ALT, "-"), + REF_VCF = case_when( + is_deletion ~ paste0(REF, substring(ALT, 2)), + TRUE ~ REF + ), + ALT_VCF = case_when( + is_insertion ~ paste0(REF, substring(ALT, 2)), + is_deletion ~ REF, + TRUE ~ ALT + ) + ) %>% + select(-is_insertion, -is_deletion) log_info("Reading annotation table") annotation <- read_tsv( @@ -42,7 +56,12 @@ log_info("Merging tables") merged <- left_join( variants, annotation, - by = c("REGION" = "CHROM", "POS", "REF", "ALT") + by = c( + "REGION" = "CHROM", + "POS" = "POS", + "REF_VCF" = "REF", + "ALT_VCF" = "ALT" + ) ) log_info("Saving results") From d0afff5fa52767b3610e127f867209c5e8b26e85 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 9 Sep 2025 16:44:01 +0200 Subject: [PATCH 063/138] Shorten heatmap columns Keeps the first annotation with an ellipsis for those with more than one annotation, to help visualization --- template.qmd | 10 +++++----- workflow/scripts/report/heatmap.R | 2 ++ 2 files changed, 7 insertions(+), 5 deletions(-) diff --git a/template.qmd b/template.qmd index 8228e97..84fed59 100644 --- a/template.qmd +++ b/template.qmd @@ -84,10 +84,10 @@ table <- read.csv(params$table) n.samples <- table %>% pull(Sample) %>% unique() %>% length() # Heatmap -vcf <- read_csv(params$heat_tab) -row.names(vcf) <- vcf$`...1` -vcf <- vcf[-1] -cor.mat <- cor(vcf, method = params$cor_method) +heatmap.df <- read_csv(params$heat_tab) +row.names(heatmap.df) <- heatmap.df$`...1` +heatmap.df <- heatmap.df[-1] +cor.mat <- cor(heatmap.df, method = params$cor_method) cor.mat[is.na(cor.mat)] <- 0 if (params$cor_method == "pearson") { cor.method.name <- "Pearson's" @@ -279,7 +279,7 @@ heatmaply_cor( cor.mat, fontsize_row = 8, fontsize_col = 8, - column_text_angle = 90 + column_text_angle = 45 ) ``` diff --git a/workflow/scripts/report/heatmap.R b/workflow/scripts/report/heatmap.R index bfcdde7..5955a75 100644 --- a/workflow/scripts/report/heatmap.R +++ b/workflow/scripts/report/heatmap.R @@ -29,6 +29,8 @@ vcf <- vcf %>% values_fn = sum ) %>% arrange(factor(SAMPLE, levels = date_order)) %>% + # Removes "|"-separated annotations, keeping the first one + ellipsis (clarifies heatmap) + rename_with(~ str_replace(., "^([^|]+)\\|.*$", "\\1(...)"), -SAMPLE) %>% column_to_rownames(var = "SAMPLE") log_info("Saving table to create heatmap") From efccc3444bd73cfb64d08039016881de78d94b17 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 9 Sep 2025 16:48:34 +0200 Subject: [PATCH 064/138] Fix typographic error --- workflow/scripts/filter_tsv.R | 2 +- workflow/scripts/report/NV_description.R | 6 +++--- workflow/scripts/report/evo_plots.R | 10 +++++----- 3 files changed, 9 insertions(+), 9 deletions(-) diff --git a/workflow/scripts/filter_tsv.R b/workflow/scripts/filter_tsv.R index d190bce..5fc1373 100644 --- a/workflow/scripts/filter_tsv.R +++ b/workflow/scripts/filter_tsv.R @@ -34,7 +34,7 @@ log_info("Finding synonymous and non synonymous variants") # Adding synonymous variable data <- mutate( data, - synonimous = case_when( + SYNONYMOUS = case_when( REF_AA == ALT_AA ~ "Yes", TRUE ~ "No" ) diff --git a/workflow/scripts/report/NV_description.R b/workflow/scripts/report/NV_description.R index 047a512..d1e4b3e 100644 --- a/workflow/scripts/report/NV_description.R +++ b/workflow/scripts/report/NV_description.R @@ -43,7 +43,7 @@ empty_vcf <- tibble( VARIANT_NAME = as.character(NA), ALT_FREQ = as.numeric(NA), GB_FEATURE = as.character(NA), - synonimous = as.character(NA), + SYNONYMOUS = as.character(NA), POS = as.numeric(NA), ALT = as.character(NA), NV_class = as.character(NA), @@ -58,7 +58,7 @@ vcf <- vcf %>% VARIANT_NAME, ALT_FREQ, GB_FEATURE, - synonimous, + SYNONYMOUS, POS, ALT ) @@ -94,7 +94,7 @@ vcf <- vcf %>% ), Class = case_when( GB_FEATURE == "Intergenic" ~ "Intergenic", - TRUE ~ synonimous + TRUE ~ SYNONYMOUS ), POS = as.numeric(POS) ) %>% diff --git a/workflow/scripts/report/evo_plots.R b/workflow/scripts/report/evo_plots.R index 9e1e1db..83b945a 100644 --- a/workflow/scripts/report/evo_plots.R +++ b/workflow/scripts/report/evo_plots.R @@ -26,7 +26,7 @@ vcf <- vcf %>% REGION, ALT_FREQ, GB_FEATURE, - synonimous, + SYNONYMOUS, POS ) @@ -45,12 +45,12 @@ vcf <- left_join(vcf, metadata) # PLOT log_info("Ploting dN and dS over time") plot_df <- vcf %>% - group_by(SAMPLE, synonimous) %>% + group_by(SAMPLE, SYNONYMOUS) %>% summarise( Freq = sum(ALT_FREQ, na.rm = TRUE) ) %>% pivot_wider( - names_from = synonimous, + names_from = SYNONYMOUS, values_from = Freq, values_fill = 0 ) %>% @@ -135,12 +135,12 @@ ggsave( # PLOT TABLES log_info("Saving plot table") vcf %>% - group_by(SAMPLE, synonimous) %>% + group_by(SAMPLE, SYNONYMOUS) %>% summarise( Freq = sum(ALT_FREQ, na.rm = TRUE) ) %>% pivot_wider( - names_from = synonimous, + names_from = SYNONYMOUS, values_from = Freq, values_fill = 0 ) %>% From 2b1b23f8e26e52fb1702187f66555adae3d248dd Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 9 Sep 2025 17:20:35 +0200 Subject: [PATCH 065/138] Detect intergenic variants using SnpEff results --- workflow/rules/vaf.smk | 1 + workflow/scripts/merge_annotation.R | 5 ++++- workflow/scripts/report/NV_description.R | 11 +++++------ workflow/scripts/report/evo_plots.R | 1 - 4 files changed, 10 insertions(+), 8 deletions(-) diff --git a/workflow/rules/vaf.smk b/workflow/rules/vaf.smk index 53f8a8c..3c9f871 100644 --- a/workflow/rules/vaf.smk +++ b/workflow/rules/vaf.smk @@ -198,6 +198,7 @@ rule merge_annotation: conda: "../envs/renv.yaml" params: ref_name = config["ALIGNMENT_REFERENCE"], + snpeff_columns = config["ANNOTATION"]["SNPEFF_COLS"], input: tsv = OUTDIR/"vaf"/"{sample}.masked.prefiltered.tsv", annot = OUTDIR/"vaf"/"{sample}.vcf_fields.longer.tsv", diff --git a/workflow/scripts/merge_annotation.R b/workflow/scripts/merge_annotation.R index 0d77f1d..5d5bbc0 100644 --- a/workflow/scripts/merge_annotation.R +++ b/workflow/scripts/merge_annotation.R @@ -38,7 +38,10 @@ variants <- read_tsv( log_info("Reading annotation table") annotation <- read_tsv( snakemake@input$annot, - col_select = c("CHROM", "POS", "REF", "ALT", "VARIANT_NAME"), + col_select = c( + snakemake@params$snpeff_columns, + "VARIANT_NAME" + ), col_types = list( CHROM = col_character(), POS = col_integer(), diff --git a/workflow/scripts/report/NV_description.R b/workflow/scripts/report/NV_description.R index d1e4b3e..3f821c1 100644 --- a/workflow/scripts/report/NV_description.R +++ b/workflow/scripts/report/NV_description.R @@ -42,7 +42,7 @@ empty_vcf <- tibble( REGION = as.character(NA), VARIANT_NAME = as.character(NA), ALT_FREQ = as.numeric(NA), - GB_FEATURE = as.character(NA), + EFFECT = as.character(NA), SYNONYMOUS = as.character(NA), POS = as.numeric(NA), ALT = as.character(NA), @@ -57,7 +57,7 @@ vcf <- vcf %>% REGION, VARIANT_NAME, ALT_FREQ, - GB_FEATURE, + EFFECT, SYNONYMOUS, POS, ALT @@ -93,7 +93,7 @@ vcf <- vcf %>% TRUE ~ "SNP" ), Class = case_when( - GB_FEATURE == "Intergenic" ~ "Intergenic", + EFFECT == "intergenic_region" ~ "Intergenic", TRUE ~ SYNONYMOUS ), POS = as.numeric(POS) @@ -104,7 +104,7 @@ vcf <- vcf %>% NV_class == "INDEL" ~ str_length(ALT) - 1 ), indel_class = case_when( - GB_FEATURE == "Intergenic" ~ "Intergenic", + EFFECT == "intergenic_region" ~ "Intergenic", NV_class == "INDEL" & indel_len %% 3 == 0 ~ "In frame", @@ -116,7 +116,7 @@ vcf <- vcf %>% ungroup() %>% mutate( group = case_when( - GB_FEATURE == "Intergenic" ~ "Intergenic", + EFFECT == "intergenic_region" ~ "Intergenic", NV_class == "SNP" ~ Class, NV_class == "INDEL" ~ indel_class ) @@ -446,7 +446,6 @@ window %>% # Heterozygous sites per sample table vcf_snp %>% filter(ALT_FREQ <= snakemake@params$max_alt_freq) %>% - select(!GB_FEATURE) %>% left_join( metadata, by = c("SAMPLE" = "ID") diff --git a/workflow/scripts/report/evo_plots.R b/workflow/scripts/report/evo_plots.R index 83b945a..f6e926b 100644 --- a/workflow/scripts/report/evo_plots.R +++ b/workflow/scripts/report/evo_plots.R @@ -25,7 +25,6 @@ vcf <- vcf %>% VARIANT_NAME, REGION, ALT_FREQ, - GB_FEATURE, SYNONYMOUS, POS ) From fd6428f7782da1bfc2f8905136a5b5360fe99962 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 9 Sep 2025 17:21:39 +0200 Subject: [PATCH 066/138] Fix Snakemake param type inside R --- workflow/scripts/merge_annotation.R | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/workflow/scripts/merge_annotation.R b/workflow/scripts/merge_annotation.R index 5d5bbc0..c5d41ba 100644 --- a/workflow/scripts/merge_annotation.R +++ b/workflow/scripts/merge_annotation.R @@ -39,7 +39,7 @@ log_info("Reading annotation table") annotation <- read_tsv( snakemake@input$annot, col_select = c( - snakemake@params$snpeff_columns, + unlist(snakemake@params$snpeff_columns), "VARIANT_NAME" ), col_types = list( From f2098ba6642697419e96060443fa8930edb91712 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 9 Sep 2025 17:23:46 +0200 Subject: [PATCH 067/138] Fix Snakemake param --- workflow/rules/vaf.smk | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/workflow/rules/vaf.smk b/workflow/rules/vaf.smk index 3c9f871..09ed0da 100644 --- a/workflow/rules/vaf.smk +++ b/workflow/rules/vaf.smk @@ -198,7 +198,7 @@ rule merge_annotation: conda: "../envs/renv.yaml" params: ref_name = config["ALIGNMENT_REFERENCE"], - snpeff_columns = config["ANNOTATION"]["SNPEFF_COLS"], + snpeff_columns = config["ANNOTATION"]["SNPEFF_COLS"].keys(), input: tsv = OUTDIR/"vaf"/"{sample}.masked.prefiltered.tsv", annot = OUTDIR/"vaf"/"{sample}.vcf_fields.longer.tsv", From 3ff2ed078f24f61e15a0312e7be14f3ea63a8ce0 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 9 Sep 2025 17:36:25 +0200 Subject: [PATCH 068/138] Restrict columns for final variant table to those that make variants unique --- config/config.yaml | 20 +++++++++++++++----- workflow/rules/vaf.smk | 2 +- workflow/scripts/merge_annotation.R | 2 +- 3 files changed, 17 insertions(+), 7 deletions(-) diff --git a/config/config.yaml b/config/config.yaml index 8fc7441..a0910c1 100644 --- a/config/config.yaml +++ b/config/config.yaml @@ -27,15 +27,15 @@ ANNOTATION: POS: POS REF: REF ALT: ALT - EFFECT: "ANN[*].EFFECT" - IMPACT: "ANN[*].IMPACT" + EFFECT: "ANN[*].EFFECT" # hard-coded column + IMPACT: "ANN[*].IMPACT" # hard-coded column BIOTYPE: "ANN[*].BIOTYPE" - GENE: "ANN[*].GENE" + GENE: "ANN[*].GENE" # hard-coded column GENEID: "ANN[*].GENEID" FEATURE: "ANN[*].FEATURE" FEATUREID: "ANN[*].FEATUREID" - HGVS_P: "ANN[*].HGVS_P" - HGVS_C: "ANN[*].HGVS_C" + HGVS_P: "ANN[*].HGVS_P" # hard-coded column + HGVS_C: "ANN[*].HGVS_C" # hard-coded column ERRORS: "ANN[*].ERRORS" # hard-coded column FILTER_INCLUDE: # IMPACT: [HIGH, MODERATE, LOW] @@ -51,6 +51,16 @@ ANNOTATION: - WARNING_TRANSCRIPT_NO_START_CODON - WARNING_TRANSCRIPT_NO_STOP_CODON VARIANT_NAME_PATTERN: "{GENE}:{coalesce(HGVS_P, HGVS_C)}" # dplyr's coalesce finds the first non-missing element + SELECT_COLS: + - CHROM + - POS + - REF + - ALT + - EFFECT + - IMPACT + - GENE + - HGVS_P + - HGVS_C DEMIX: PATHOGEN: "SARS-CoV-2" MIN_QUALITY: 20 diff --git a/workflow/rules/vaf.smk b/workflow/rules/vaf.smk index 09ed0da..ef53627 100644 --- a/workflow/rules/vaf.smk +++ b/workflow/rules/vaf.smk @@ -198,7 +198,7 @@ rule merge_annotation: conda: "../envs/renv.yaml" params: ref_name = config["ALIGNMENT_REFERENCE"], - snpeff_columns = config["ANNOTATION"]["SNPEFF_COLS"].keys(), + select_columns = config["ANNOTATION"]["SELECT_COLS"], input: tsv = OUTDIR/"vaf"/"{sample}.masked.prefiltered.tsv", annot = OUTDIR/"vaf"/"{sample}.vcf_fields.longer.tsv", diff --git a/workflow/scripts/merge_annotation.R b/workflow/scripts/merge_annotation.R index c5d41ba..8b80452 100644 --- a/workflow/scripts/merge_annotation.R +++ b/workflow/scripts/merge_annotation.R @@ -39,7 +39,7 @@ log_info("Reading annotation table") annotation <- read_tsv( snakemake@input$annot, col_select = c( - unlist(snakemake@params$snpeff_columns), + unlist(snakemake@params$select_cols), "VARIANT_NAME" ), col_types = list( From b54c88e1504cdac570d69ff42a2f2c86373887ab Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 9 Sep 2025 17:39:25 +0200 Subject: [PATCH 069/138] Fix param name --- workflow/scripts/merge_annotation.R | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/workflow/scripts/merge_annotation.R b/workflow/scripts/merge_annotation.R index 8b80452..11a8024 100644 --- a/workflow/scripts/merge_annotation.R +++ b/workflow/scripts/merge_annotation.R @@ -39,7 +39,7 @@ log_info("Reading annotation table") annotation <- read_tsv( snakemake@input$annot, col_select = c( - unlist(snakemake@params$select_cols), + unlist(snakemake@params$select_columns), "VARIANT_NAME" ), col_types = list( From d81601f88859eed19e248282901a0ca78a32d14d Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 9 Sep 2025 17:43:22 +0200 Subject: [PATCH 070/138] Update selected columns --- config/config.yaml | 10 ---------- workflow/rules/vaf.smk | 1 - workflow/scripts/merge_annotation.R | 7 ++++++- 3 files changed, 6 insertions(+), 12 deletions(-) diff --git a/config/config.yaml b/config/config.yaml index a0910c1..4d16753 100644 --- a/config/config.yaml +++ b/config/config.yaml @@ -51,16 +51,6 @@ ANNOTATION: - WARNING_TRANSCRIPT_NO_START_CODON - WARNING_TRANSCRIPT_NO_STOP_CODON VARIANT_NAME_PATTERN: "{GENE}:{coalesce(HGVS_P, HGVS_C)}" # dplyr's coalesce finds the first non-missing element - SELECT_COLS: - - CHROM - - POS - - REF - - ALT - - EFFECT - - IMPACT - - GENE - - HGVS_P - - HGVS_C DEMIX: PATHOGEN: "SARS-CoV-2" MIN_QUALITY: 20 diff --git a/workflow/rules/vaf.smk b/workflow/rules/vaf.smk index ef53627..53f8a8c 100644 --- a/workflow/rules/vaf.smk +++ b/workflow/rules/vaf.smk @@ -198,7 +198,6 @@ rule merge_annotation: conda: "../envs/renv.yaml" params: ref_name = config["ALIGNMENT_REFERENCE"], - select_columns = config["ANNOTATION"]["SELECT_COLS"], input: tsv = OUTDIR/"vaf"/"{sample}.masked.prefiltered.tsv", annot = OUTDIR/"vaf"/"{sample}.vcf_fields.longer.tsv", diff --git a/workflow/scripts/merge_annotation.R b/workflow/scripts/merge_annotation.R index 11a8024..fb9dcf5 100644 --- a/workflow/scripts/merge_annotation.R +++ b/workflow/scripts/merge_annotation.R @@ -39,7 +39,12 @@ log_info("Reading annotation table") annotation <- read_tsv( snakemake@input$annot, col_select = c( - unlist(snakemake@params$select_columns), + "CHROM", + "POS", + "REF", + "ALT", + "EFFECT", + "IMPACT", "VARIANT_NAME" ), col_types = list( From 23e3cff254ab2fbacd612db26042b008c5b76d8a Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 9 Sep 2025 17:56:02 +0200 Subject: [PATCH 071/138] Remove intermediate per-site annotation table --- workflow/rules/vaf.smk | 19 +----------- workflow/scripts/filter_tsv.R | 7 ----- workflow/scripts/report/get_annotation.py | 36 ----------------------- 3 files changed, 1 insertion(+), 61 deletions(-) delete mode 100644 workflow/scripts/report/get_annotation.py diff --git a/workflow/rules/vaf.smk b/workflow/rules/vaf.smk index 53f8a8c..6253941 100644 --- a/workflow/rules/vaf.smk +++ b/workflow/rules/vaf.smk @@ -52,22 +52,6 @@ rule snps_to_ancestor: """ -rule annotation: - threads:1 - conda: "../envs/biopython.yaml" - shadow: "shallow" - input: - gb = OUTDIR/"reference.gb", - ref = OUTDIR/"reference.fasta", - features = Path(config["FEATURES_JSON"]).resolve() - output: - df = temp(OUTDIR/"annotation.csv") - log: - LOGDIR / "annotation" / "log.txt" - script: - "../scripts/report/get_annotation.py" - - rule mask_tsv: threads: 1 conda: "../envs/biopython.yaml" @@ -92,8 +76,7 @@ rule filter_tsv: min_alt_rv = 2, min_alt_dp = 2, input: - tsv = OUTDIR/"vaf"/"{sample}.masked.tsv", - annotation = OUTDIR/"annotation.csv" + tsv = OUTDIR/"vaf"/"{sample}.masked.tsv" output: filtered_tsv = temp(OUTDIR/"vaf"/"{sample}.masked.prefiltered.tsv") log: diff --git a/workflow/scripts/filter_tsv.R b/workflow/scripts/filter_tsv.R index 5fc1373..69801e1 100644 --- a/workflow/scripts/filter_tsv.R +++ b/workflow/scripts/filter_tsv.R @@ -43,13 +43,6 @@ data <- mutate( # Remove duplicated features data <- distinct(data, pick(!GFF_FEATURE), .keep_all = TRUE) -# Change annotation to gb2seq annotation -features <- read_csv(snakemake@input[["annotation"]]) - -data <- data %>% - left_join(features) %>% - rename(GB_FEATURE = GEN) - log_info("Saving results") write_tsv( data, diff --git a/workflow/scripts/report/get_annotation.py b/workflow/scripts/report/get_annotation.py deleted file mode 100644 index 552d70d..0000000 --- a/workflow/scripts/report/get_annotation.py +++ /dev/null @@ -1,36 +0,0 @@ -#!/usr/bin/env python3 - -import json -import logging -import pandas as pd -from gb2seq.features import Features -from Bio import SeqIO - - -def main(): - logging.basicConfig(filename=snakemake.log[0], format=snakemake.config["LOG_PY_FMT"], level=logging.INFO) - - logging.info("Reading features") - ft = Features(snakemake.input.gb) - with open(snakemake.input.features) as f: - feature_key = json.load(f) - - logging.info("Reading reference") - reference = str(next(SeqIO.parse(snakemake.input.ref, "fasta")).seq) - positions = [x for x in range(len(reference))] - - logging.info("Building annotation") - genes = [] - for pos in positions: - if len(ft.getFeatureNames(pos)) == 0: - genes.append("Intergenic") - else: - genes.append(feature_key[list(ft.getFeatureNames(pos))[0]]) - - logging.info("Writing table") - df = pd.DataFrame({"POS": positions, "GEN": genes}) - df.to_csv(snakemake.output.df, index= False) - - -if __name__ == "__main__": - main() From 7b8218be2442f12057d5d6893827a1d06f1c43a5 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 9 Sep 2025 18:05:08 +0200 Subject: [PATCH 072/138] Use text instead of labels for pseudovolcano plot --- workflow/scripts/report/snp_plots.R | 8 ++++++-- 1 file changed, 6 insertions(+), 2 deletions(-) diff --git a/workflow/scripts/report/snp_plots.R b/workflow/scripts/report/snp_plots.R index be2f7ba..34a95ee 100644 --- a/workflow/scripts/report/snp_plots.R +++ b/workflow/scripts/report/snp_plots.R @@ -52,7 +52,11 @@ vcf <- vcf %>% # Fill positions without alt frequency with 0 vcf <- vcf %>% - complete(nesting(REGION, VARIANT_NAME, POS), SAMPLE, fill = list(ALT_FREQ = 0)) + complete( + nesting(REGION, VARIANT_NAME, POS), + SAMPLE, + fill = list(ALT_FREQ = 0) + ) # Join variants file and metadata file vcf <- left_join(vcf, data, by = c("SAMPLE" = "ID")) @@ -128,7 +132,7 @@ volcano <- cor.df.fill %>% y = trans.p ) + geom_point() + - geom_label_repel(aes(label = label), max.overlaps = 1000) + + geom_text_repel(aes(label = label), max.overlaps = 1000, direction = "x") + xlim(c(-1, 1)) + geom_hline( aes(yintercept = -log10(0.05)), From 9b47abe7ad94f765aad1262793bfe67999a6fab1 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Thu, 11 Sep 2025 16:41:00 +0200 Subject: [PATCH 073/138] Move features in JSON file to config YAML --- config/config.yaml | 15 ++++++- config/sarscov2_features.json | 14 ------ workflow/rules/evolution.smk | 7 +-- workflow/rules/report.smk | 6 +-- workflow/scripts/N_S_sites_from_fasta.py | 46 ++++++++++++-------- workflow/scripts/report/NV_description.R | 23 +++++----- workflow/scripts/report/evo_plots.R | 2 +- workflow/scripts/window.py | 55 ++++++++++++++---------- 8 files changed, 92 insertions(+), 76 deletions(-) delete mode 100644 config/sarscov2_features.json diff --git a/config/config.yaml b/config/config.yaml index 4d16753..8640575 100644 --- a/config/config.yaml +++ b/config/config.yaml @@ -2,8 +2,6 @@ ALIGNMENT_REFERENCE: "NC_045512.2" PROBLEMATIC_VCF: "https://raw.githubusercontent.com/W-L/ProblematicSites_SARS-CoV2/da322c32004f7b16bdaa6a8ee7fd24d56e79d9dc/problematic_sites_sarsCov2.vcf" -FEATURES_JSON: - "config/sarscov2_features.json" COORDINATES_JSON: "config/sarscov2_coordinates.json" GENETIC_CODE_JSON: @@ -65,6 +63,19 @@ DEMIX: WINDOW: WIDTH: 1000 STEP: 50 +GB_FEATURES: + "ORF1ab polyprotein": "ORF1ab" + "ORF1a polyprotein": "ORF1ab" + "surface glycoprotein": "S" + "ORF3a protein": "ORF3a" + "envelope protein": "E" + "membrane glycoprotein": "M" + "ORF6 protein": "ORF6" + "ORF7a protein": "ORF7" + "ORF7b": "ORF7" + "ORF8 protein": "ORF8" + "nucleocapsid phosphoprotein": "N" + "ORF10 protein": "ORF10" GISAID: CREDENTIALS: "config/gisaid.yaml" DATE_COLUMN: "CollectionDate" diff --git a/config/sarscov2_features.json b/config/sarscov2_features.json deleted file mode 100644 index 2031f32..0000000 --- a/config/sarscov2_features.json +++ /dev/null @@ -1,14 +0,0 @@ -{ - "ORF1ab polyprotein": "orf1ab", - "ORF1a polyprotein": "orf1ab", - "surface glycoprotein": "S", - "ORF3a protein": "ORF3a", - "envelope protein": "E", - "membrane glycoprotein": "M", - "ORF6 protein": "ORF6", - "ORF7a protein": "ORF7", - "ORF7b": "ORF7", - "ORF8 protein": "ORF8", - "nucleocapsid phosphoprotein": "N", - "ORF10 protein": "ORF10" -} diff --git a/workflow/rules/evolution.smk b/workflow/rules/evolution.smk index e89d358..6c9908c 100644 --- a/workflow/rules/evolution.smk +++ b/workflow/rules/evolution.smk @@ -1,13 +1,14 @@ rule N_S_sites: threads: 1 conda: "../envs/biopython.yaml" + params: + gb_features = config["GB_FEATURES"], input: fasta = OUTDIR/f"{OUTPUT_NAME}.ancestor.fasta", gb = OUTDIR/"reference.gb", - features = Path(config["FEATURES_JSON"]).resolve(), - genetic_code = Path(config["GENETIC_CODE_JSON"]).resolve() + genetic_code = Path(config["GENETIC_CODE_JSON"]).resolve(), output: - csv = temp(OUTDIR/f"{OUTPUT_NAME}.ancestor.N_S.sites.csv") + csv = temp(OUTDIR/f"{OUTPUT_NAME}.ancestor.N_S.sites.csv"), log: LOGDIR / "N_S_sites" / "log.txt" script: diff --git a/workflow/rules/report.smk b/workflow/rules/report.smk index 707ceab..64504c5 100644 --- a/workflow/rules/report.smk +++ b/workflow/rules/report.smk @@ -44,11 +44,11 @@ rule window: conda: "../envs/biopython.yaml" params: window = config["WINDOW"]["WIDTH"], - step = config["WINDOW"]["STEP"] + step = config["WINDOW"]["STEP"], + select_gb_features = config.get("GB_FEATURES", {}), # if empty, uses all available features input: vcf = OUTDIR/f"{OUTPUT_NAME}.variants.tsv", gb = OUTDIR/"reference.gb", - features = config["FEATURES_JSON"] output: window_df = temp(OUTDIR/f"{OUTPUT_NAME}.window.csv"), log: @@ -229,7 +229,7 @@ rule evo_plots: params: design = config["PLOTS"] input: - N_S = OUTDIR/f"{OUTPUT_NAME}.ancestor.N_S.sites.csv", + n_s_sites = OUTDIR/f"{OUTPUT_NAME}.ancestor.N_S.sites.csv", vcf = OUTDIR/f"{OUTPUT_NAME}.variants.tsv", metadata = config["METADATA"] output: diff --git a/workflow/scripts/N_S_sites_from_fasta.py b/workflow/scripts/N_S_sites_from_fasta.py index e98ac94..e03de22 100644 --- a/workflow/scripts/N_S_sites_from_fasta.py +++ b/workflow/scripts/N_S_sites_from_fasta.py @@ -11,7 +11,9 @@ def split_into_codons(seq: str) -> list: """Split the complete CDS feature in to a list of codons""" - codons = [seq[i:i + 3] for i in range(0, len(seq), 3) if "N" not in seq[i:i + 3]] + codons = [ + seq[i:i + 3] for i in range(0, len(seq), 3) if "N" not in seq[i:i + 3] + ] return codons @@ -29,7 +31,7 @@ def calculate_potential_changes(genetic_code: dict) -> dict: for codon_p in range(0, 3): nts = ["A", "G", "T", "C"] # Do not consider self substitutions, e.g. A->A - nts.remove(codon[codon_p]) + nts.remove(codon[codon_p]) for nt in nts: codon_mutated = list(codon) # Mutate the basepair @@ -43,49 +45,55 @@ def calculate_potential_changes(genetic_code: dict) -> dict: def get_feature_codons(alignment: Gb2Alignment, annotation: list) -> dict: - dct = {key:alignment.ntSequences(key)[1].sequence for key in annotation} - return {key:split_into_codons(item) for key,item in dct.items()} + dct = {key: alignment.ntSequences(key)[1].sequence for key in annotation} + return {key: split_into_codons(item) for key, item in dct.items()} -def get_df(codons: dict, genetic_code: dict) -> pd.DataFrame: - keys = [] +def calculate_ns_sites(codons: dict, genetic_code: dict) -> pd.DataFrame: + features = [] N_sites = [] S_sites = [] values = calculate_potential_changes(genetic_code) for key, item in codons.items(): - keys.append(key) + features.append(key) N = sum([values["N"][x] for x in item if x in values["N"].keys()]) S = sum([values["S"][x] for x in item if x in values["S"].keys()]) N_sites.append(N) - S_sites.append(S) - return pd.DataFrame({"gene": keys, "N": N_sites, "S": S_sites}) + S_sites.append(S) + return pd.DataFrame({"gene": features, "N": N_sites, "S": S_sites}) def main(): - logging.basicConfig(filename=snakemake.log[0], format=snakemake.config["LOG_PY_FMT"], level=logging.INFO) - + logging.basicConfig( + filename=snakemake.log[0], format=snakemake.config["LOG_PY_FMT"], + level=logging.INFO + ) + logging.info("Reading features") - with open(snakemake.input.features) as f: - feature_list = list(json.load(f).keys()) + feature_list = list(snakemake.params.gb_features.keys()) logging.info("Reading genetic code") with open(snakemake.input.genetic_code) as f: genetic_code = json.load(f) - + logging.info("Create alignment object") features = Features(snakemake.input.gb) - seq = list(FastaReads(snakemake.input.fasta))[0] + fasta_reads = list(FastaReads(snakemake.input.fasta)) + if len(fasta_reads) > 1: + logging.warning( + f"More than one record found in {snakemake.input.fasta}, selecting the first one") + seq = fasta_reads[0] aln = Gb2Alignment(seq, features) - logging.info("Splitting ancestral sequence into codons") - codons_dict = get_feature_codons(aln, feature_list) + logging.info("Splitting input sequence into codons") + codons = get_feature_codons(aln, feature_list) logging.info("Calculating synonymous and non synonymous sites") - df = get_df(codons_dict, genetic_code) + df = calculate_ns_sites(codons, genetic_code) logging.info("Saving results") - df.to_csv(snakemake.output.csv,index= False) + df.to_csv(snakemake.output.csv, index=False) if __name__ == "__main__": diff --git a/workflow/scripts/report/NV_description.R b/workflow/scripts/report/NV_description.R index 3f821c1..5e9ffd1 100644 --- a/workflow/scripts/report/NV_description.R +++ b/workflow/scripts/report/NV_description.R @@ -198,8 +198,8 @@ window_plot <- window %>% ggplot() + aes( x = position, - y = fractions, - color = gen + y = fraction, + color = feature ) + geom_point() + geom_line( @@ -209,7 +209,7 @@ window_plot <- window %>% ) + scale_y_continuous( label = scales::percent, - limits = c(0, max(window$fractions) + 0.005) + limits = c(0, max(window$fraction) + 0.005) ) + xlim(c(0, 29903)) + scale_color_manual(values = GENE_PALETTE) + @@ -235,7 +235,7 @@ window_plot_nsp <- window_plot + data = npc, aes( x = (summary_start + summary_end) / 2, - y = max(window$fractions) + 0.002, + y = max(window$fraction) + 0.002, label = summary_nsp ), inherit.aes = FALSE, @@ -266,9 +266,8 @@ ggsave( # Zoom in in spike log_info("Plotting summary for variants in the spike") - spike_pos <- window %>% - filter(gen == "S") %>% + filter(feature == "S") %>% pull(position) vcf_spike <- vcf %>% @@ -278,12 +277,12 @@ vcf_spike <- vcf %>% ) window_plot_spike <- window %>% - filter(gen == "S") %>% + filter(feature == "S") %>% ggplot() + aes( x = position, - y = fractions, - color = gen + y = fraction, + color = feature ) + geom_point() + geom_line( @@ -293,7 +292,7 @@ window_plot_spike <- window %>% ) + scale_y_continuous( label = scales::percent, - limits = c(0, max(window$fractions) + 0.005) + limits = c(0, max(window$fraction) + 0.005) ) + xlim(c(min(spike_pos), max(spike_pos))) + scale_color_manual( @@ -438,8 +437,8 @@ vcf %>% window %>% transmute( POS = position, - feature = gen, - prop_PolymorphicSites = fractions + feature = feature, + prop_PolymorphicSites = fraction ) %>% write.csv(snakemake@output[["table_1"]], row.names = FALSE) diff --git a/workflow/scripts/report/evo_plots.R b/workflow/scripts/report/evo_plots.R index f6e926b..5eebc00 100644 --- a/workflow/scripts/report/evo_plots.R +++ b/workflow/scripts/report/evo_plots.R @@ -15,7 +15,7 @@ source(snakemake@params[["design"]]) # Read inputs vcf <- read_delim(snakemake@input[["vcf"]]) metadata <- read_delim(snakemake@input[["metadata"]]) -N_S_position <- read_delim(snakemake@input[["N_S"]]) +N_S_position <- read_delim(snakemake@input[["n_s_sites"]]) # DATA PROCESSING # Create SNP variable and select useful variables diff --git a/workflow/scripts/window.py b/workflow/scripts/window.py index 5ba6a8c..4dfd281 100644 --- a/workflow/scripts/window.py +++ b/workflow/scripts/window.py @@ -6,46 +6,57 @@ from gb2seq.features import Features -def window_calculation(sites: set, window: int, step: int, coord: str) -> pd.DataFrame: - ft = Features(coord) # Create Features object to obtain annotations - positions = [] - pct = [] - genes = [] - lim_sup = len(ft.reference.sequence) + 1 +def window_calculation(sites: set, window: int, step: int, gb_features: Features) -> pd.DataFrame: + positions, fractions, features = [], [], [] + lim_sup = len(gb_features.reference.sequence) + 1 for position in range(1, lim_sup, step): - if len(ft.getFeatureNames(position)) == 0: - genes.append("Intergenic") + if len(gb_features.getFeatureNames(position)) == 0: + features.append("Intergenic") else: - genes.append(list(ft.getFeatureNames(position))[0]) + # If more than one feature on a site, include both (sorted lexicographically) + features.append("|".join(sorted(gb_features.getFeatureNames(position)))) # Add percent (excluding initial and final positions) if position - window not in range(1, lim_sup): - pct.append(0) + fractions.append(0.0) else: # Calculate no. of polimorphisms in the window - num_snp = len([x for x in sites if x in range(position - window, position + 1)]) - pct.append(num_snp / window) + num_snp = len([x for x in sites if x in range( + position - window, position + 1)]) + fractions.append(num_snp / window) positions.append(position) - return pd.DataFrame({"position": positions, "fractions": pct, "gen": genes}) + return pd.DataFrame({"position": positions, "fraction": fractions, "feature": features}) def main(): - logging.basicConfig(filename=snakemake.log[0], format=snakemake.config["LOG_PY_FMT"], level=logging.INFO) + logging.basicConfig( + filename=snakemake.log[0], format=snakemake.config["LOG_PY_FMT"], + level=logging.INFO + ) logging.info("Reading input VCF") df = pd.read_table(snakemake.input.vcf) sites = set(df.POS) - logging.info("Reading features") - with open(snakemake.input.features) as f: - features_key = json.load(f) + logging.info("Reading genbank features") + features = Features(snakemake.input.gb) + + logging.info("Calculating polimorphic sites sliding window") + windows = window_calculation( + sites, + snakemake.params.window, + snakemake.params.step, + features + ) + + if len(snakemake.params.select_gb_features) != 0: + logging.info("Filtering and renaming genbank features") + windows = windows[ + windows.feature.isin(snakemake.params.select_gb_features.keys()) + ].replace(snakemake.params.select_gb_features) - logging.info("Sliding window calculation of proportion of polimorphic sites") - frame = window_calculation(sites, snakemake.params.window, snakemake.params.step, snakemake.input.gb) - logging.info("Saving results") - frame.replace(features_key, inplace = True) - frame.to_csv(snakemake.output.window_df, index= False) + windows.to_csv(snakemake.output.window_df, index=False) if __name__ == "__main__": From e75a9fd2ff23423559ae6dbc6f2f3700cd7a5470 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Thu, 11 Sep 2025 16:55:15 +0200 Subject: [PATCH 074/138] Remove genbank feature --- config/config.yaml | 1 - 1 file changed, 1 deletion(-) diff --git a/config/config.yaml b/config/config.yaml index 8640575..41ce4b5 100644 --- a/config/config.yaml +++ b/config/config.yaml @@ -65,7 +65,6 @@ WINDOW: STEP: 50 GB_FEATURES: "ORF1ab polyprotein": "ORF1ab" - "ORF1a polyprotein": "ORF1ab" "surface glycoprotein": "S" "ORF3a protein": "ORF3a" "envelope protein": "E" From 29f944a9ad8608023ec69d1445fadde772681b11 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Thu, 11 Sep 2025 17:24:05 +0200 Subject: [PATCH 075/138] Fix filtering and formatting --- workflow/scripts/window.py | 37 +++++++++++++++++++++++++++++-------- 1 file changed, 29 insertions(+), 8 deletions(-) diff --git a/workflow/scripts/window.py b/workflow/scripts/window.py index 4dfd281..f055ed5 100644 --- a/workflow/scripts/window.py +++ b/workflow/scripts/window.py @@ -13,20 +13,40 @@ def window_calculation(sites: set, window: int, step: int, gb_features: Features if len(gb_features.getFeatureNames(position)) == 0: features.append("Intergenic") else: - # If more than one feature on a site, include both (sorted lexicographically) - features.append("|".join(sorted(gb_features.getFeatureNames(position)))) + # Include all features on site + features.append(gb_features.getFeatureNames(position)) # Add percent (excluding initial and final positions) if position - window not in range(1, lim_sup): fractions.append(0.0) else: # Calculate no. of polimorphisms in the window - num_snp = len([x for x in sites if x in range( - position - window, position + 1)]) + num_snp = sum( + 1 for x in sites if x in range(position - window, position + 1) + ) fractions.append(num_snp / window) positions.append(position) return pd.DataFrame({"position": positions, "fraction": fractions, "feature": features}) +def select_and_format_features(window_features: set) -> str | None: + selected_features = sorted( + snakemake.params.select_gb_features[feat] + if feat in snakemake.params.select_gb_features + for feat in window_features + ) + if len(selected_features) != 0: + return "|".join(selected_features) + else: + return None + + +def format_features(window_features: set) -> str: + if len(window_features) != 0: + return "|".join(sorted(window_features)) + else: + return None + + def main(): logging.basicConfig( @@ -50,10 +70,11 @@ def main(): ) if len(snakemake.params.select_gb_features) != 0: - logging.info("Filtering and renaming genbank features") - windows = windows[ - windows.feature.isin(snakemake.params.select_gb_features.keys()) - ].replace(snakemake.params.select_gb_features) + logging.info("Selecting and formatting genbank features") + windows.feature = windows.feature.map(select_and_format_features) + else: + logging.info("Formatting genbank features") + windows.feature = windows.feature.map(format_features) logging.info("Saving results") windows.to_csv(snakemake.output.window_df, index=False) From 34cf7bc82379ac4395996bb20b3e3e0bde03c8de Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Thu, 11 Sep 2025 17:24:46 +0200 Subject: [PATCH 076/138] Fix syntax error --- workflow/scripts/window.py | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/workflow/scripts/window.py b/workflow/scripts/window.py index f055ed5..b1e8b81 100644 --- a/workflow/scripts/window.py +++ b/workflow/scripts/window.py @@ -31,8 +31,8 @@ def window_calculation(sites: set, window: int, step: int, gb_features: Features def select_and_format_features(window_features: set) -> str | None: selected_features = sorted( snakemake.params.select_gb_features[feat] - if feat in snakemake.params.select_gb_features for feat in window_features + if feat in snakemake.params.select_gb_features ) if len(selected_features) != 0: return "|".join(selected_features) From 241382931e1f157be61b1692e35a9c075682f873 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Thu, 11 Sep 2025 17:27:14 +0200 Subject: [PATCH 077/138] Update ORF1ab color --- config/design_plots.R | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/config/design_plots.R b/config/design_plots.R index 166577f..1fb0e7f 100644 --- a/config/design_plots.R +++ b/config/design_plots.R @@ -29,7 +29,7 @@ theme_update( GENE_PALETTE <- c( M = "#B4D4B4", N = "#B7B7B8", - orf1ab = "#9CC4DC", + ORF1ab = "#9CC4DC", ORF3a = "#ECB4B7", ORF8 = "#996D2B", S = "#F5CC9E", From 1e426f1424a0078ae11619a4a8f58d4ee3371010 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Thu, 11 Sep 2025 17:31:37 +0200 Subject: [PATCH 078/138] Update ORF1ab name --- config/sarscov2_coordinates.json | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/config/sarscov2_coordinates.json b/config/sarscov2_coordinates.json index 9626dce..a7242f5 100644 --- a/config/sarscov2_coordinates.json +++ b/config/sarscov2_coordinates.json @@ -1,6 +1,6 @@ { "five_prime_UTR": [1, 265], - "orf1ab": [266, 21555], + "ORF1ab": [266, 21555], "Intergenic_1": [21556, 21562], "S": [21563, 25384], "Intergenic_2": [25385, 25392], From 08c5c4dbdaeb11cdd7f078b5c8c2ec3849f72776 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Thu, 11 Sep 2025 17:45:09 +0200 Subject: [PATCH 079/138] Avoid repeated features --- workflow/scripts/window.py | 8 +++++--- 1 file changed, 5 insertions(+), 3 deletions(-) diff --git a/workflow/scripts/window.py b/workflow/scripts/window.py index b1e8b81..5ceb3fa 100644 --- a/workflow/scripts/window.py +++ b/workflow/scripts/window.py @@ -30,9 +30,11 @@ def window_calculation(sites: set, window: int, step: int, gb_features: Features def select_and_format_features(window_features: set) -> str | None: selected_features = sorted( - snakemake.params.select_gb_features[feat] - for feat in window_features - if feat in snakemake.params.select_gb_features + set( + snakemake.params.select_gb_features[feat] + for feat in window_features + if feat in snakemake.params.select_gb_features + ) ) if len(selected_features) != 0: return "|".join(selected_features) From 0b2e08e1fecee8f6373b76e973d9dd1bdfdd9cb8 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Thu, 16 Oct 2025 12:05:44 +0200 Subject: [PATCH 080/138] Update dN/dS calculation Adds flexibility and fixes potential issues related to extraction of genome features --- config/config.yaml | 25 +++--- workflow/rules/evolution.smk | 3 +- workflow/scripts/N_S_sites_from_fasta.py | 105 +++++++++++++++-------- workflow/scripts/report/evo_plots.R | 8 +- 4 files changed, 91 insertions(+), 50 deletions(-) diff --git a/config/config.yaml b/config/config.yaml index 41ce4b5..c69a5fd 100644 --- a/config/config.yaml +++ b/config/config.yaml @@ -64,17 +64,20 @@ WINDOW: WIDTH: 1000 STEP: 50 GB_FEATURES: - "ORF1ab polyprotein": "ORF1ab" - "surface glycoprotein": "S" - "ORF3a protein": "ORF3a" - "envelope protein": "E" - "membrane glycoprotein": "M" - "ORF6 protein": "ORF6" - "ORF7a protein": "ORF7" - "ORF7b": "ORF7" - "ORF8 protein": "ORF8" - "nucleocapsid phosphoprotein": "N" - "ORF10 protein": "ORF10" + include: # any + product: + - "ORF1ab polyprotein" + - "surface glycoprotein" + - "ORF3a protein" + - "envelope protein" + - "membrane glycoprotein" + - "ORF6 protein" + - "ORF7a protein" + - "ORF7b" + - "ORF8 protein" + - "nucleocapsid phosphoprotein" + - "ORF10 protein" + # exclude: ... # all GISAID: CREDENTIALS: "config/gisaid.yaml" DATE_COLUMN: "CollectionDate" diff --git a/workflow/rules/evolution.smk b/workflow/rules/evolution.smk index 6c9908c..5bc41f5 100644 --- a/workflow/rules/evolution.smk +++ b/workflow/rules/evolution.smk @@ -2,7 +2,8 @@ rule N_S_sites: threads: 1 conda: "../envs/biopython.yaml" params: - gb_features = config["GB_FEATURES"], + features = config.get("GB_FEATURES", {}), + gb_qualifier_display = "gene", input: fasta = OUTDIR/f"{OUTPUT_NAME}.ancestor.fasta", gb = OUTDIR/"reference.gb", diff --git a/workflow/scripts/N_S_sites_from_fasta.py b/workflow/scripts/N_S_sites_from_fasta.py index e03de22..0668c84 100644 --- a/workflow/scripts/N_S_sites_from_fasta.py +++ b/workflow/scripts/N_S_sites_from_fasta.py @@ -3,33 +3,40 @@ import logging import json import itertools as it +from typing import Dict + import pandas as pd -from gb2seq.alignment import Gb2Alignment -from gb2seq.features import Features -from dark.fasta import FastaReads +from Bio import SeqIO +from Bio.SeqRecord import SeqRecord +from Bio.Seq import Seq + + +NTS = ("A", "C", "G", "T") -def split_into_codons(seq: str) -> list: +def read_monofasta(path: str) -> SeqRecord: + return SeqIO.read(path, format="fasta") + + +def split_into_codons(seq: Seq) -> list: """Split the complete CDS feature in to a list of codons""" - codons = [ - seq[i:i + 3] for i in range(0, len(seq), 3) if "N" not in seq[i:i + 3] + return [ + seq[i:i + 3] for i in range(0, len(seq)-2, 3) if all(char in NTS for char in seq[i:i + 3]) ] - return codons def calculate_potential_changes(genetic_code: dict) -> dict: """Generate a dictionary with S and N pre-calculated for all possible codons""" # Initialize structure - nts = set(["A", "G", "T", "C"]) potential_changes = {"S": {}, "N": {}} - for codon in it.product(nts, repeat=3): - potential_changes["S"]["".join(codon)] = 0. - potential_changes["N"]["".join(codon)] = 0. + for codon in it.product(NTS, repeat=3): + potential_changes["S"]["".join(codon)] = 0.0 + potential_changes["N"]["".join(codon)] = 0.0 # Mutate (substitutions) all possible codons in the given genetic code # and count proportions of mutations that are synonymous and non-synonmyous for codon in genetic_code.keys(): for codon_p in range(0, 3): - nts = ["A", "G", "T", "C"] + nts = list(NTS) # Do not consider self substitutions, e.g. A->A nts.remove(codon[codon_p]) for nt in nts: @@ -38,21 +45,19 @@ def calculate_potential_changes(genetic_code: dict) -> dict: codon_mutated[codon_p] = nt # Count how many of them are synonymous if genetic_code[codon] == genetic_code["".join(codon_mutated)]: - potential_changes["S"][codon] += 1/3. + potential_changes["S"][codon] += 1 / 3.0 else: - potential_changes["N"][codon] += 1/3. + potential_changes["N"][codon] += 1 / 3.0 return potential_changes -def get_feature_codons(alignment: Gb2Alignment, annotation: list) -> dict: - dct = {key: alignment.ntSequences(key)[1].sequence for key in annotation} - return {key: split_into_codons(item) for key, item in dct.items()} +def get_feature_codons(coding_records: Dict[str, SeqRecord]) -> dict: + return {name: split_into_codons(record.seq) for name, record in coding_records.items()} def calculate_ns_sites(codons: dict, genetic_code: dict) -> pd.DataFrame: features = [] - N_sites = [] - S_sites = [] + N_sites, S_sites = [], [] values = calculate_potential_changes(genetic_code) for key, item in codons.items(): features.append(key) @@ -60,7 +65,7 @@ def calculate_ns_sites(codons: dict, genetic_code: dict) -> pd.DataFrame: S = sum([values["S"][x] for x in item if x in values["S"].keys()]) N_sites.append(N) S_sites.append(S) - return pd.DataFrame({"gene": features, "N": N_sites, "S": S_sites}) + return pd.DataFrame({"feature": features, "N": N_sites, "S": S_sites}) def main(): @@ -70,24 +75,56 @@ def main(): level=logging.INFO ) - logging.info("Reading features") - feature_list = list(snakemake.params.gb_features.keys()) - logging.info("Reading genetic code") with open(snakemake.input.genetic_code) as f: genetic_code = json.load(f) - logging.info("Create alignment object") - features = Features(snakemake.input.gb) - fasta_reads = list(FastaReads(snakemake.input.fasta)) - if len(fasta_reads) > 1: - logging.warning( - f"More than one record found in {snakemake.input.fasta}, selecting the first one") - seq = fasta_reads[0] - aln = Gb2Alignment(seq, features) - - logging.info("Splitting input sequence into codons") - codons = get_feature_codons(aln, feature_list) + logging.info("Reading GenBank file") + gb = SeqIO.read(snakemake.input.gb, format="gb") + + logging.info("Reading input FASTA") + record = read_monofasta(snakemake.input.fasta) + + if len(snakemake.params.features) == 0: + logging.debug("Selecting all features") + feature_iterator = iter(gb.features) + else: + included = snakemake.params.features.get("include", {}) + excluded = snakemake.params.features.get("exclude", {}) + logging.debug(f"Selecting features including any of {included} and excluding all of {excluded}") + feature_iterator = ( + feature for feature in gb.features + if any( + (qualifier_value in included.get(qualifier_key, [])) + for qualifier_key in included.keys() + for qualifier_value in feature.qualifiers.get(qualifier_key, []) + ) and all( + (qualifier_value not in excluded.get(qualifier_key, [])) + for qualifier_key in excluded.keys() + for qualifier_value in feature.qualifiers.get(qualifier_key, []) + ) + ) + + logging.info("Extracting CDS") + coding_records = {} + for feature in feature_iterator: + logging.debug( + "Processing SeqFeature: " + f"ID={feature.id} type={feature.type} location={feature.location} " + f"gene={feature.qualifiers.get('gene', [])} " + f"locus_tag={feature.qualifiers.get('locus_tag', [])} " + f"product={feature.qualifiers.get('product', [])}" + ) + identifier = "|".join(feature.qualifiers.get(snakemake.params.gb_qualifier_display, [])) + if identifier == "": + logging.error(f"Feature at {feature.location} has no qualifier '{snakemake.params.gb_qualifier_display}'") + elif identifier in coding_records: + logging.warning(f"Identifier '{identifier}' is already among coding records and will not be replaced by feature at {feature.location}") + else: + coding_records[identifier] = feature.extract(record) + + logging.info(f"Splitting {len(coding_records)} records into codons") + codons = get_feature_codons(coding_records) logging.info("Calculating synonymous and non synonymous sites") df = calculate_ns_sites(codons, genetic_code) diff --git a/workflow/scripts/report/evo_plots.R b/workflow/scripts/report/evo_plots.R index 5eebc00..fd9f348 100644 --- a/workflow/scripts/report/evo_plots.R +++ b/workflow/scripts/report/evo_plots.R @@ -81,19 +81,19 @@ plot <- plot_df %>% geom_point(size = 3) + geom_line() + scale_color_manual( - name = "Parameter", + name = "", labels = DNDS_LABELS, values = DNDS_COLORS ) + scale_shape_manual( - name = "Parameter", + name = "", values = DNDS_SHAPES, labels = DNDS_LABELS ) + labs( y = "Substitution rate", x = "Days since the initial sampling", - color = "Parameter" + color = "" ) ggsave( @@ -119,7 +119,7 @@ plot_omega <- plot_df %>% labs( y = "w (dN/dS)", x = "Days since the initial sampling", - color = "Parameter" + color = "" ) ggsave( From c2c5b04292b4e3889deca44c5f0dab0957c4f477 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Thu, 16 Oct 2025 14:18:01 +0200 Subject: [PATCH 081/138] Use genbank qualifiers for window annotation --- workflow/rules/report.smk | 5 +- workflow/scripts/window.py | 104 +++++++++++++++++++++++-------------- 2 files changed, 67 insertions(+), 42 deletions(-) diff --git a/workflow/rules/report.smk b/workflow/rules/report.smk index 64504c5..7fe8c97 100644 --- a/workflow/rules/report.smk +++ b/workflow/rules/report.smk @@ -45,9 +45,10 @@ rule window: params: window = config["WINDOW"]["WIDTH"], step = config["WINDOW"]["STEP"], - select_gb_features = config.get("GB_FEATURES", {}), # if empty, uses all available features + features = config.get("GB_FEATURES", {}), + gb_qualifier_display = "gene" input: - vcf = OUTDIR/f"{OUTPUT_NAME}.variants.tsv", + variants = OUTDIR/f"{OUTPUT_NAME}.variants.tsv", gb = OUTDIR/"reference.gb", output: window_df = temp(OUTDIR/f"{OUTPUT_NAME}.window.csv"), diff --git a/workflow/scripts/window.py b/workflow/scripts/window.py index 5ceb3fa..c7f5632 100644 --- a/workflow/scripts/window.py +++ b/workflow/scripts/window.py @@ -1,25 +1,51 @@ #!/usr/bin/env python3 import logging -import json import pandas as pd -from gb2seq.features import Features +from typing import NewType, Dict, Iterable, List +from Bio import SeqIO +from Bio.SeqFeature import SeqFeature +from Bio.SeqFeature import CompoundLocation, FeatureLocation -def window_calculation(sites: set, window: int, step: int, gb_features: Features) -> pd.DataFrame: + +FeatureIndex = NewType("FeatureIndex", Dict[str, FeatureLocation | CompoundLocation]) + + +def index_features(features: Iterable[SeqFeature]) -> FeatureIndex: + index = FeatureIndex({}) + for feature in features: + identifier = "|".join(feature.qualifiers.get(snakemake.params.gb_qualifier_display, [])) + if identifier == "": + logging.error(f"Feature at {feature.location} has no qualifier '{snakemake.params.gb_qualifier_display}' and will be skipped") + elif feature.location is None: + logging.warning(f"Feature '{identifier}' has no location and will be skipped") + elif identifier in index: + logging.warning(f"Identifier '{identifier}' is already in feature index and will not be replaced by feature at {feature.location}") + else: + index[identifier] = feature.location + return index + + +def feature_names_at(position: int, feature_index: FeatureIndex) -> List[str]: + return [name for name, location in feature_index.items() if position in location] + + +def window_calculation(sites: set, window: int, step: int, size: int, feature_index: FeatureIndex) -> pd.DataFrame: positions, fractions, features = [], [], [] - lim_sup = len(gb_features.reference.sequence) + 1 + lim_sup = size + 1 for position in range(1, lim_sup, step): - if len(gb_features.getFeatureNames(position)) == 0: + feature_names = ";".join(feature_names_at(position, feature_index)) + if len(feature_names) == 0: features.append("Intergenic") else: # Include all features on site - features.append(gb_features.getFeatureNames(position)) + features.append(feature_names) # Add percent (excluding initial and final positions) if position - window not in range(1, lim_sup): fractions.append(0.0) else: - # Calculate no. of polimorphisms in the window + # Calculate number of polymorphisms in the window num_snp = sum( 1 for x in sites if x in range(position - window, position + 1) ) @@ -28,27 +54,6 @@ def window_calculation(sites: set, window: int, step: int, gb_features: Features return pd.DataFrame({"position": positions, "fraction": fractions, "feature": features}) -def select_and_format_features(window_features: set) -> str | None: - selected_features = sorted( - set( - snakemake.params.select_gb_features[feat] - for feat in window_features - if feat in snakemake.params.select_gb_features - ) - ) - if len(selected_features) != 0: - return "|".join(selected_features) - else: - return None - - -def format_features(window_features: set) -> str: - if len(window_features) != 0: - return "|".join(sorted(window_features)) - else: - return None - - def main(): logging.basicConfig( @@ -56,28 +61,47 @@ def main(): level=logging.INFO ) - logging.info("Reading input VCF") - df = pd.read_table(snakemake.input.vcf) + # Read input files + logging.info("Reading input variants file") + df = pd.read_table(snakemake.input.variants) sites = set(df.POS) - logging.info("Reading genbank features") - features = Features(snakemake.input.gb) + logging.info("Reading GenBank file") + gb = SeqIO.read(snakemake.input.gb, format="gb") + + # Build feature iterator given the provided filters + if len(snakemake.params.features) == 0: + logging.debug("Selecting all features") + feature_iterator = iter(gb.features) + else: + included = snakemake.params.features.get("include", {}) + excluded = snakemake.params.features.get("exclude", {}) + logging.debug(f"Selecting features including any of {included} and excluding all of {excluded}") + feature_iterator = ( + feature for feature in gb.features + if any( + (qualifier_value in included.get(qualifier_key, [])) + for qualifier_key in included.keys() + for qualifier_value in feature.qualifiers.get(qualifier_key, []) + ) and all( + (qualifier_value not in excluded.get(qualifier_key, [])) + for qualifier_key in excluded.keys() + for qualifier_value in feature.qualifiers.get(qualifier_key, []) + ) + ) + + logging.info("Indexing feature locations") + feature_index = index_features(feature_iterator) logging.info("Calculating polimorphic sites sliding window") windows = window_calculation( sites, snakemake.params.window, snakemake.params.step, - features + len(gb), + feature_index ) - if len(snakemake.params.select_gb_features) != 0: - logging.info("Selecting and formatting genbank features") - windows.feature = windows.feature.map(select_and_format_features) - else: - logging.info("Formatting genbank features") - windows.feature = windows.feature.map(format_features) - logging.info("Saving results") windows.to_csv(snakemake.output.window_df, index=False) From aa743decb7a3260b959f0d9d9b6c1a1dc467f70e Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Thu, 16 Oct 2025 14:18:28 +0200 Subject: [PATCH 082/138] Remove unused dependency Avoids depending on gb2seq, using BioPython instead --- workflow/envs/biopython.yaml | 4 ---- 1 file changed, 4 deletions(-) diff --git a/workflow/envs/biopython.yaml b/workflow/envs/biopython.yaml index 172f7bf..aaafd09 100644 --- a/workflow/envs/biopython.yaml +++ b/workflow/envs/biopython.yaml @@ -5,7 +5,3 @@ dependencies: - python==3.10 - biopython==1.81 - pandas==2.0.3 - - pip==23.2.1 - - mafft==7.525 - - pip: - - gb2seq==0.2.20 From cc995fa12d08023b53578119cbce06899dfed378 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Thu, 16 Oct 2025 15:09:39 +0200 Subject: [PATCH 083/138] Update config key case for genbank features --- config/config.yaml | 4 ++-- workflow/scripts/N_S_sites_from_fasta.py | 4 ++-- workflow/scripts/window.py | 4 ++-- 3 files changed, 6 insertions(+), 6 deletions(-) diff --git a/config/config.yaml b/config/config.yaml index c69a5fd..74f8bce 100644 --- a/config/config.yaml +++ b/config/config.yaml @@ -64,7 +64,7 @@ WINDOW: WIDTH: 1000 STEP: 50 GB_FEATURES: - include: # any + INCLUDE: # any product: - "ORF1ab polyprotein" - "surface glycoprotein" @@ -77,7 +77,7 @@ GB_FEATURES: - "ORF8 protein" - "nucleocapsid phosphoprotein" - "ORF10 protein" - # exclude: ... # all + # EXCLUDE: ... # all GISAID: CREDENTIALS: "config/gisaid.yaml" DATE_COLUMN: "CollectionDate" diff --git a/workflow/scripts/N_S_sites_from_fasta.py b/workflow/scripts/N_S_sites_from_fasta.py index 0668c84..4978ed2 100644 --- a/workflow/scripts/N_S_sites_from_fasta.py +++ b/workflow/scripts/N_S_sites_from_fasta.py @@ -89,8 +89,8 @@ def main(): logging.debug("Selecting all features") feature_iterator = iter(gb.features) else: - included = snakemake.params.features.get("include", {}) - excluded = snakemake.params.features.get("exclude", {}) + included = snakemake.params.features.get("INCLUDE", {}) + excluded = snakemake.params.features.get("EXCLUDE", {}) logging.debug(f"Selecting features including any of {included} and excluding all of {excluded}") feature_iterator = ( feature for feature in gb.features diff --git a/workflow/scripts/window.py b/workflow/scripts/window.py index c7f5632..b2a7a43 100644 --- a/workflow/scripts/window.py +++ b/workflow/scripts/window.py @@ -74,8 +74,8 @@ def main(): logging.debug("Selecting all features") feature_iterator = iter(gb.features) else: - included = snakemake.params.features.get("include", {}) - excluded = snakemake.params.features.get("exclude", {}) + included = snakemake.params.features.get("INCLUDE", {}) + excluded = snakemake.params.features.get("EXCLUDE", {}) logging.debug(f"Selecting features including any of {included} and excluding all of {excluded}") feature_iterator = ( feature for feature in gb.features From 5c6cfd569b08c96b02bff07b8366f31adc42799b Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Thu, 16 Oct 2025 15:10:45 +0200 Subject: [PATCH 084/138] Remove unused entry from test config file --- .test/targets.yaml | 2 -- 1 file changed, 2 deletions(-) diff --git a/.test/targets.yaml b/.test/targets.yaml index 73c05fb..652a5fb 100644 --- a/.test/targets.yaml +++ b/.test/targets.yaml @@ -40,8 +40,6 @@ PLOT_GENOME_REGIONS: "config/nsp_annotation.csv" REPORT_QMD: "template.qmd" -FEATURES_JSON: - "config/sarscov2_features.json" COORDINATES_JSON: "config/sarscov2_coordinates.json" GENETIC_CODE_JSON: From f80f75b4f14cf2c16dbce5b2f761185f1aff82ce Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Thu, 16 Oct 2025 16:32:58 +0200 Subject: [PATCH 085/138] Handle unset or empty GenBank feature filters Clarifies feature iteration and filtering, and performs "exclude" filter after the "include" filter --- workflow/scripts/N_S_sites_from_fasta.py | 76 +++++++++++++++------- workflow/scripts/window.py | 82 ++++++++++++++++-------- 2 files changed, 110 insertions(+), 48 deletions(-) diff --git a/workflow/scripts/N_S_sites_from_fasta.py b/workflow/scripts/N_S_sites_from_fasta.py index 4978ed2..97cd10d 100644 --- a/workflow/scripts/N_S_sites_from_fasta.py +++ b/workflow/scripts/N_S_sites_from_fasta.py @@ -3,11 +3,12 @@ import logging import json import itertools as it -from typing import Dict +from typing import Dict, Iterable import pandas as pd from Bio import SeqIO from Bio.SeqRecord import SeqRecord +from Bio.SeqFeature import SeqFeature from Bio.Seq import Seq @@ -68,6 +69,54 @@ def calculate_ns_sites(codons: dict, genetic_code: dict) -> pd.DataFrame: return pd.DataFrame({"feature": features, "N": N_sites, "S": S_sites}) +def iter_features_filtering(features: Iterable[SeqFeature], included: Dict[str, str], excluded: Dict[str, str]) -> Iterable[SeqFeature]: + # No filters + if len(included) == 0 and len(excluded) == 0: + logging.debug("Selecting all features") + return iter(features) + # Only inclusion filter + elif len(included) == 0 and len(excluded) != 0: + logging.debug(f"Selecting features excluding all of {excluded}") + return ( + feature for feature in features + if all( + (qualifier_value not in excluded.get(qualifier_key, [])) + for qualifier_key in excluded.keys() + for qualifier_value in feature.qualifiers.get(qualifier_key, []) + ) + ) + # Only exclusion filter + elif len(included) != 0 and len(excluded) == 0: + logging.debug(f"Selecting features including any of {included}") + return ( + feature for feature in features + if any( + (qualifier_value in included.get(qualifier_key, [])) + for qualifier_key in included.keys() + for qualifier_value in feature.qualifiers.get(qualifier_key, []) + ) + ) + # Inclusion then exclusion filter + else: + logging.debug(f"Selecting features including any of {included} and then excluding all of {excluded}") + included_features = ( + feature for feature in features + if any( + (qualifier_value in included.get(qualifier_key, [])) + for qualifier_key in included.keys() + for qualifier_value in feature.qualifiers.get(qualifier_key, []) + ) + ) + return ( + feature for feature in included_features + if all( + (qualifier_value not in excluded.get(qualifier_key, [])) + for qualifier_key in excluded.keys() + for qualifier_value in feature.qualifiers.get(qualifier_key, []) + ) + ) + + def main(): logging.basicConfig( @@ -85,29 +134,11 @@ def main(): logging.info("Reading input FASTA") record = read_monofasta(snakemake.input.fasta) - if len(snakemake.params.features) == 0: - logging.debug("Selecting all features") - feature_iterator = iter(gb.features) - else: - included = snakemake.params.features.get("INCLUDE", {}) - excluded = snakemake.params.features.get("EXCLUDE", {}) - logging.debug(f"Selecting features including any of {included} and excluding all of {excluded}") - feature_iterator = ( - feature for feature in gb.features - if any( - (qualifier_value in included.get(qualifier_key, [])) - for qualifier_key in included.keys() - for qualifier_value in feature.qualifiers.get(qualifier_key, []) - ) and all( - (qualifier_value not in excluded.get(qualifier_key, [])) - for qualifier_key in excluded.keys() - for qualifier_value in feature.qualifiers.get(qualifier_key, []) - ) - ) - logging.info("Extracting CDS") coding_records = {} - for feature in feature_iterator: + included = snakemake.params.features.get("INCLUDE", {}) + excluded = snakemake.params.features.get("EXCLUDE", {}) + for feature in iter_features_filtering(gb.features, included, excluded): logging.debug( "Processing SeqFeature: " f"ID={feature.id} type={feature.type} location={feature.location} " @@ -121,6 +152,7 @@ def main(): elif identifier in coding_records: logging.warning(f"Identifier '{identifier}' is already among coding records and will not be replaced by feature at {feature.location}") else: + logging.debug(f"Adding feature") coding_records[identifier] = feature.extract(record) logging.info(f"Splitting {len(coding_records)} records into codons") diff --git a/workflow/scripts/window.py b/workflow/scripts/window.py index b2a7a43..f5633ed 100644 --- a/workflow/scripts/window.py +++ b/workflow/scripts/window.py @@ -1,15 +1,62 @@ #!/usr/bin/env python3 import logging -import pandas as pd from typing import NewType, Dict, Iterable, List +import pandas as pd from Bio import SeqIO -from Bio.SeqFeature import SeqFeature -from Bio.SeqFeature import CompoundLocation, FeatureLocation +from Bio.SeqFeature import SeqFeature, SimpleLocation, CompoundLocation -FeatureIndex = NewType("FeatureIndex", Dict[str, FeatureLocation | CompoundLocation]) +FeatureIndex = NewType("FeatureIndex", Dict[str, SimpleLocation | CompoundLocation]) + + +def iter_features_filtering(features: Iterable[SeqFeature], included: Dict[str, str], excluded: Dict[str, str]) -> Iterable[SeqFeature]: + # No filters + if len(included) == 0 and len(excluded) == 0: + logging.debug("Selecting all features") + return iter(features) + # Only inclusion filter + elif len(included) == 0 and len(excluded) != 0: + logging.debug(f"Selecting features excluding all of {excluded}") + return ( + feature for feature in features + if all( + (qualifier_value not in excluded.get(qualifier_key, [])) + for qualifier_key in excluded.keys() + for qualifier_value in feature.qualifiers.get(qualifier_key, []) + ) + ) + # Only exclusion filter + elif len(included) != 0 and len(excluded) == 0: + logging.debug(f"Selecting features including any of {included}") + return ( + feature for feature in features + if any( + (qualifier_value in included.get(qualifier_key, [])) + for qualifier_key in included.keys() + for qualifier_value in feature.qualifiers.get(qualifier_key, []) + ) + ) + # Inclusion then exclusion filter + else: + logging.debug(f"Selecting features including any of {included} and then excluding all of {excluded}") + included_features = ( + feature for feature in features + if any( + (qualifier_value in included.get(qualifier_key, [])) + for qualifier_key in included.keys() + for qualifier_value in feature.qualifiers.get(qualifier_key, []) + ) + ) + return ( + feature for feature in included_features + if all( + (qualifier_value not in excluded.get(qualifier_key, [])) + for qualifier_key in excluded.keys() + for qualifier_value in feature.qualifiers.get(qualifier_key, []) + ) + ) def index_features(features: Iterable[SeqFeature]) -> FeatureIndex: @@ -69,29 +116,12 @@ def main(): logging.info("Reading GenBank file") gb = SeqIO.read(snakemake.input.gb, format="gb") - # Build feature iterator given the provided filters - if len(snakemake.params.features) == 0: - logging.debug("Selecting all features") - feature_iterator = iter(gb.features) - else: - included = snakemake.params.features.get("INCLUDE", {}) - excluded = snakemake.params.features.get("EXCLUDE", {}) - logging.debug(f"Selecting features including any of {included} and excluding all of {excluded}") - feature_iterator = ( - feature for feature in gb.features - if any( - (qualifier_value in included.get(qualifier_key, [])) - for qualifier_key in included.keys() - for qualifier_value in feature.qualifiers.get(qualifier_key, []) - ) and all( - (qualifier_value not in excluded.get(qualifier_key, [])) - for qualifier_key in excluded.keys() - for qualifier_value in feature.qualifiers.get(qualifier_key, []) - ) - ) - logging.info("Indexing feature locations") - feature_index = index_features(feature_iterator) + included = snakemake.params.features.get("INCLUDE", {}) + excluded = snakemake.params.features.get("EXCLUDE", {}) + feature_index = index_features( + iter_features_filtering(gb.features, included, excluded) + ) logging.info("Calculating polimorphic sites sliding window") windows = window_calculation( From b613ef23d8346904fb59f1ada5e953292669a257 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Thu, 16 Oct 2025 16:35:56 +0200 Subject: [PATCH 086/138] Document GB_FEATURES config key --- config/README.md | 16 +++++++++++++++- 1 file changed, 15 insertions(+), 1 deletion(-) diff --git a/config/README.md b/config/README.md index 1e97134..564dbd5 100644 --- a/config/README.md +++ b/config/README.md @@ -130,7 +130,6 @@ All of the following variables are pre-defined in [config.yaml](/config/config.y - `ALIGNMENT_REFERENCE`: NCBI accession number of the reference record for sequence alignment. - `PROBLEMATIC_VCF`: URL or path of a VCF file containing problematic genome positions for masking. -- `FEATURES_JSON`: path of a JSON file containing name equivalences of genome features for data visualization. - `COORDINATES_JSON`: path of a JSON file containing genome feature coordinates for data visualization. - `GENETIC_CODE_JSON`: path of a JSON file containing a genetic code for gene translation. - `TREE_MODEL`: substitution model used by IQTREE (see [docs](http://www.iqtree.org/doc/Substitution-Models)). @@ -155,6 +154,18 @@ All of the following variables are pre-defined in [config.yaml](/config/config.y - `WINDOW`: sliding window of nucleotide variants per site configuration: - `WIDTH`: number of sites within windows. - `STEP`: number of sites between windows. +- `GB_FEATURES`: optional mapping to filter which features from the GenBank file are used + by some analyses (e.g. rules `window` and `N_S_sites`). + If `GB_FEATURES` is empty or unset, all features are used. + Filtering is applied in order: + - `INCLUDE`: mapping of qualifier names to sequences of values. + If present, only features that match at least one key/value pair in `INCLUDE` + are included in the analyses. For example, having `INCLUDE: {gene: [S, N]}` keeps features whose `gene` + qualifier equals `S` or `N`. + - `EXCLUDE`: mapping of qualifier names to sequences of values. + After `INCLUDE` is applied, any feature that matches any key/value pair in `EXCLUDE` + is omitted. For example, having `EXCLUDE: {gene: [S, N]}` removes features whose + `gene` qualifier equals `S` or `N`. - `GISAID`: automatic context download configuration. - `CREDENTIALS`: path of the GISAID credentials in YAML format. - `DATE_COLUMN`: name of the column that contains sampling dates (YYYY-MM-DD) in the input target metadata. @@ -170,6 +181,9 @@ All of the following variables are pre-defined in [config.yaml](/config/config.y - `PLOT_GENOME_REGIONS`: path of a CSV file containing genome regions, e.g. SARS-CoV-2 non-structural protein (NSP) coordinates, for data visualization. - `REPORT_QMD`: path of the report template in Quarto markdown (QMD) format. +TODO: describe new entries in config.yaml +TODO: describe issues with many cores (rules freyja and snps_to_ancestor) + ## Workflow graphs To generate a simplified rule graph, run: From 2b420c9d09a1f8bc5cd6e8a3ca5b9bde02f2adb3 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Fri, 17 Oct 2025 10:37:51 +0200 Subject: [PATCH 087/138] Decouple dN/dS plots from data --- config/design_plots.R | 8 +- workflow/rules/evolution.smk | 14 +++ workflow/rules/report.smk | 17 ++- workflow/scripts/dnds_data.R | 68 ++++++++++++ workflow/scripts/report/dnds_plots.R | 85 ++++++++++++++ workflow/scripts/report/evo_plots.R | 159 --------------------------- 6 files changed, 178 insertions(+), 173 deletions(-) create mode 100644 workflow/scripts/dnds_data.R create mode 100644 workflow/scripts/report/dnds_plots.R delete mode 100644 workflow/scripts/report/evo_plots.R diff --git a/config/design_plots.R b/config/design_plots.R index 1fb0e7f..86460f3 100644 --- a/config/design_plots.R +++ b/config/design_plots.R @@ -93,11 +93,11 @@ DNDS_LABELS <- c( ) DNDS_COLORS <- c( - dn = "#E53E47", - ds = "#2C47F5" + dN = "#E53E47", + dS = "#2C47F5" ) DNDS_SHAPES <- c( - dn = 2, - ds = 4 + dN = 2, + dS = 4 ) diff --git a/workflow/rules/evolution.smk b/workflow/rules/evolution.smk index 5bc41f5..6edcb97 100644 --- a/workflow/rules/evolution.smk +++ b/workflow/rules/evolution.smk @@ -14,3 +14,17 @@ rule N_S_sites: LOGDIR / "N_S_sites" / "log.txt" script: "../scripts/N_S_sites_from_fasta.py" + + +rule dnds_data: + conda: "../envs/renv.yaml" + input: + n_s_sites = OUTDIR/f"{OUTPUT_NAME}.ancestor.N_S.sites.csv", + variants = OUTDIR/f"{OUTPUT_NAME}.variants.tsv", + metadata = config["METADATA"] + output: + table = report(REPORT_DIR_TABLES/"dnds.csv") + log: + LOGDIR / "dnds_data" / "log.txt" + script: + "../scripts/dnds_data.R" diff --git a/workflow/rules/report.smk b/workflow/rules/report.smk index 7fe8c97..1f9c60f 100644 --- a/workflow/rules/report.smk +++ b/workflow/rules/report.smk @@ -225,22 +225,19 @@ rule time_signal_plot: "../scripts/report/time_signal_plot.R" -rule evo_plots: +rule dnds_plots: conda: "../envs/renv.yaml" params: design = config["PLOTS"] input: - n_s_sites = OUTDIR/f"{OUTPUT_NAME}.ancestor.N_S.sites.csv", - vcf = OUTDIR/f"{OUTPUT_NAME}.variants.tsv", - metadata = config["METADATA"] + table = REPORT_DIR_TABLES/"dnds.csv", output: - plot = report(REPORT_DIR_PLOTS/"figure_11.png"), - plot_omega = report(REPORT_DIR_PLOTS/"figure_12.png"), - table = report(REPORT_DIR_TABLES/"figure_11.csv") + plot_dn_ds = report(REPORT_DIR_PLOTS/"dn_and_ds.png"), + plot_omega = report(REPORT_DIR_PLOTS/"dnds.png"), log: LOGDIR / "evo_plots" / "log.txt" script: - "../scripts/report/evo_plots.R" + "../scripts/report/dnds_plots.R" rule snp_plots: @@ -292,8 +289,8 @@ rule report: tree = report(REPORT_DIR_PLOTS/"allele_freq_tree.png"), temest = report(REPORT_DIR_PLOTS/"time_signal.png"), heat_table = report(REPORT_DIR_TABLES/"heatmap.csv"), - evo = report(REPORT_DIR_PLOTS/"figure_11.png"), - omega_plot = report(REPORT_DIR_PLOTS/"figure_12.png"), + evo = report(REPORT_DIR_PLOTS/"dn_and_ds.png"), + omega_plot = report(REPORT_DIR_PLOTS/"dnds.png"), freyja_ts = OUTDIR/"demixing"/"freyja_data"/"last_barcode_update.txt", value = REPORT_DIR_TABLES/"diversity.json", stats_lm = REPORT_DIR_TABLES/"time_signal.json", diff --git a/workflow/scripts/dnds_data.R b/workflow/scripts/dnds_data.R new file mode 100644 index 0000000..b71b23d --- /dev/null +++ b/workflow/scripts/dnds_data.R @@ -0,0 +1,68 @@ +#!/usr/bin/env Rscript + +# Write stdout and stderr to log file +log <- file(snakemake@log[[1]], open = "wt") +sink(log, type = "message") +sink(log, type = "output") + +library(tidyverse) +library(logger) +log_threshold(INFO) + +# Read inputs +log_info("Reading variants table") +variants <- read_delim( + snakemake@input[["variants"]], + col_select = c( + "SAMPLE", + "VARIANT_NAME", + "REGION", + "ALT_FREQ", + "SYNONYMOUS", + "POS" + ) +) + +log_info("Reading metadata table") +metadata <- read_delim(snakemake@input[["metadata"]]) %>% + mutate( + interval = as.numeric( + as.Date(CollectionDate) - min(as.Date(CollectionDate)) + ) + ) %>% + select(ID, interval) %>% + rename(SAMPLE = ID) + +log_debug("Adding metadata to variants table") +variants <- left_join(variants, metadata) + +log_info("Reading N/S sites") +N_S_position <- read_delim(snakemake@input[["n_s_sites"]]) + +log_info("Computing dN/dS over time (NG86)") +dn.ds <- variants %>% + group_by(SAMPLE, SYNONYMOUS) %>% + summarise( + Freq = sum(ALT_FREQ, na.rm = TRUE) + ) %>% + pivot_wider( + names_from = SYNONYMOUS, + values_from = Freq, + values_fill = 0 + ) %>% + transmute( + dn = No / sum(N_S_position$N), + ds = Yes / sum(N_S_position$S) + ) %>% + ungroup() %>% + left_join(unique(select(variants, SAMPLE, interval))) %>% + transmute( + sample = SAMPLE, + day = interval, + dN = dn, + dS = ds, + w = dn / ds + ) + +log_info("Writing results") +write_csv(dn.ds, snakemake@output[["table"]]) diff --git a/workflow/scripts/report/dnds_plots.R b/workflow/scripts/report/dnds_plots.R new file mode 100644 index 0000000..f3aa651 --- /dev/null +++ b/workflow/scripts/report/dnds_plots.R @@ -0,0 +1,85 @@ +#!/usr/bin/env Rscript + +# Write stdout and stderr to log file +log <- file(snakemake@log[[1]], open = "wt") +sink(log, type = "message") +sink(log, type = "output") + +library(tidyverse) +library(logger) +log_threshold(INFO) + +# Import file with plots style +source(snakemake@params[["design"]]) + +# Read inputs +log_info("Reading plot data") +plot.df <- read_delim(snakemake@input$table) %>% + filter(w != Inf) %>% + pivot_longer( + c("dN", "dS", "w"), + values_to = "value", + names_to = "ratio" + ) + +log_info("Plotting dN and dS") +p.dn.ds <- plot.df %>% + filter(ratio != "w") %>% + ggplot() + + aes( + x = day, + y = value, + color = ratio, + shape = ratio + ) + + geom_point(size = 3) + + geom_line() + + scale_color_manual( + name = "", + labels = DNDS_LABELS, + values = DNDS_COLORS + ) + + scale_shape_manual( + name = "", + values = DNDS_SHAPES, + labels = DNDS_LABELS + ) + + labs( + y = "Substitution rate", + x = "Days since the initial sampling", + color = "" + ) + +ggsave( + filename = snakemake@output$plot_dn_ds, + plot = p.dn.ds, + width = 159.2, + height = 119.4, + units = "mm", + dpi = 250 +) + +log_info("Plotting w (dN/dS)") +p.omega <- plot.df %>% + filter(ratio == "w") %>% + ggplot() + + aes( + x = day, + y = value, + ) + + geom_point(color = "black") + + geom_line(color = "black") + + labs( + y = "w (dN/dS)", + x = "Days since the initial sampling", + color = "" + ) + +ggsave( + filename = snakemake@output$plot_omega, + plot = p.omega, + width = 159.2, + height = 119.4, + units = "mm", + dpi = 250 +) diff --git a/workflow/scripts/report/evo_plots.R b/workflow/scripts/report/evo_plots.R deleted file mode 100644 index fd9f348..0000000 --- a/workflow/scripts/report/evo_plots.R +++ /dev/null @@ -1,159 +0,0 @@ -#!/usr/bin/env Rscript - -# Write stdout and stderr to log file -log <- file(snakemake@log[[1]], open = "wt") -sink(log, type = "message") -sink(log, type = "output") - -library(tidyverse) -library(logger) -log_threshold(INFO) - -# Import file with plots style -source(snakemake@params[["design"]]) - -# Read inputs -vcf <- read_delim(snakemake@input[["vcf"]]) -metadata <- read_delim(snakemake@input[["metadata"]]) -N_S_position <- read_delim(snakemake@input[["n_s_sites"]]) - -# DATA PROCESSING -# Create SNP variable and select useful variables -vcf <- vcf %>% - dplyr::select( - SAMPLE, - VARIANT_NAME, - REGION, - ALT_FREQ, - SYNONYMOUS, - POS - ) - -# Create variable for days sins first sample in metadata -metadata <- metadata %>% - mutate( - interval = as.numeric( - as.Date(CollectionDate) - min(as.Date(CollectionDate)) - ) - ) %>% - select(ID, interval) %>% - rename(SAMPLE = ID) - -vcf <- left_join(vcf, metadata) - -# PLOT -log_info("Ploting dN and dS over time") -plot_df <- vcf %>% - group_by(SAMPLE, SYNONYMOUS) %>% - summarise( - Freq = sum(ALT_FREQ, na.rm = TRUE) - ) %>% - pivot_wider( - names_from = SYNONYMOUS, - values_from = Freq, - values_fill = 0 - ) %>% - transmute( - dn = No / sum(N_S_position$N), - ds = Yes / sum(N_S_position$S) - ) %>% - ungroup() %>% - mutate( - w = dn / ds, - ) %>% - filter(w != Inf) %>% - pivot_longer( - c("dn", "ds", "w"), - values_to = "value", - names_to = "d" - ) %>% - left_join(unique(select(vcf, SAMPLE, interval))) - -plot <- plot_df %>% - filter(d != "w") %>% - ggplot() + - aes( - x = interval, - y = value, - color = d, - shape = d - ) + - geom_point(size = 3) + - geom_line() + - scale_color_manual( - name = "", - labels = DNDS_LABELS, - values = DNDS_COLORS - ) + - scale_shape_manual( - name = "", - values = DNDS_SHAPES, - labels = DNDS_LABELS - ) + - labs( - y = "Substitution rate", - x = "Days since the initial sampling", - color = "" - ) - -ggsave( - filename = snakemake@output[["plot"]], - plot = plot, - width = 159.2, - height = 119.4, - units = "mm", - dpi = 250 -) - -# Plot for omega - -plot_omega <- plot_df %>% - filter(d == "w") %>% - ggplot() + - aes( - x = interval, - y = value, - ) + - geom_point(color = "black") + - geom_line(color = "black") + - labs( - y = "w (dN/dS)", - x = "Days since the initial sampling", - color = "" - ) - -ggsave( - filename = snakemake@output[["plot_omega"]], - plot = plot_omega, - width = 159.2, - height = 119.4, - units = "mm", - dpi = 250 -) - -# PLOT TABLES -log_info("Saving plot table") -vcf %>% - group_by(SAMPLE, SYNONYMOUS) %>% - summarise( - Freq = sum(ALT_FREQ, na.rm = TRUE) - ) %>% - pivot_wider( - names_from = SYNONYMOUS, - values_from = Freq, - values_fill = 0 - ) %>% - transmute( - dn = No / sum(N_S_position$N), - ds = Yes / sum(N_S_position$S) - ) %>% - ungroup() %>% - left_join(unique(select(vcf, SAMPLE, interval))) %>% - transmute( - sample = SAMPLE, - DaysSinceFirst = interval, - dN = dn, - dS = ds, - w = dn / ds - ) %>% - write.csv(snakemake@output[["table"]], row.names = FALSE) From dd1e1ad0e1fd4f02e3cff4bced84b16ac88d5111 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Fri, 17 Oct 2025 11:52:07 +0200 Subject: [PATCH 088/138] Decouple SNP correlation (pseudovolcano) plot from data --- workflow/rules/report.smk | 86 ++++++++++++-- .../report/af_time_correlation_per_snp_data.R | 111 ++++++++++++++++++ .../report/af_time_correlation_per_snp_plot.R | 50 ++++++++ 3 files changed, 234 insertions(+), 13 deletions(-) create mode 100644 workflow/scripts/report/af_time_correlation_per_snp_data.R create mode 100644 workflow/scripts/report/af_time_correlation_per_snp_plot.R diff --git a/workflow/rules/report.smk b/workflow/rules/report.smk index 1f9c60f..3a92726 100644 --- a/workflow/rules/report.smk +++ b/workflow/rules/report.smk @@ -240,24 +240,84 @@ rule dnds_plots: "../scripts/report/dnds_plots.R" -rule snp_plots: +rule af_time_correlation_per_snp_data: conda: "../envs/renv.yaml" params: - design = config["PLOTS"], cor_method = config["COR"]["METHOD"], - cor_exact = config["COR"]["EXACT"] + cor_exact = config["COR"]["EXACT"], input: - vcf = OUTDIR/f"{OUTPUT_NAME}.variants.tsv", - metadata = config["METADATA"] + variants = OUTDIR/f"{OUTPUT_NAME}.variants.tsv", + metadata = config["METADATA"], + output: + fmt_variants = temp(OUTDIR/f"{OUTPUT_NAME}.variants.filled.dated.tsv"), + correlations = report(REPORT_DIR_TABLES/"af_time_correlation_per_snp.csv"), # fig 6 + log: + LOGDIR / "af_time_correlation_per_snp_data" / "log.txt" + script: + "../scripts/report/af_time_correlation_per_snp_data.R" + + +rule af_time_correlation_per_snp_plot: + conda: "../envs/renv.yaml" + params: + design = config["PLOTS"], + input: + correlations = REPORT_DIR_TABLES/"af_time_correlation_per_snp.csv", + output: + plot = report(REPORT_DIR_PLOTS/"af_time_correlation_per_snp.png"), # fig 6 + log: + LOGDIR / "af_time_correlation_per_snp_plot" / "log.txt" + script: + "../scripts/report/af_time_correlation_per_snp_plot.R" + + +rule af_trajectory_panel_data: # panel with AF trajectories in time + # TODO + conda: "../envs/renv.yaml" + input: + fmt_variants = OUTDIR/f"{OUTPUT_NAME}.variants.filled.dated.tsv", + correlations = REPORT_DIR_TABLES/"af_time_correlation_per_snp.csv", + output: + table = report(REPORT_DIR_TABLES/"af_trajectory_panel.csv"), # fig 7 + log: + LOGDIR / "af_trajectory_panel_data" / "log.txt" + script: + "../scripts/report/af_trajectory_panel_data.R" + + +rule af_trajectory_panel_plot: # panel with AF trajectories in time + # TODO + conda: "../envs/renv.yaml" + params: + design = config["PLOTS"], + input: + table = REPORT_DIR_TABLES/"af_trajectory_panel.csv", output: - pseudovolcano = report(REPORT_DIR_PLOTS/"figure_6.png"), - snp_panel = report(REPORT_DIR_PLOTS/"figure_7.png"), - table_1 = report(REPORT_DIR_TABLES/"figure_6.csv"), - table_2 = report(REPORT_DIR_TABLES/"figure_7.csv") + plot = report(REPORT_DIR_PLOTS/"af_trajectory_panel.png"), # fig 7 log: - LOGDIR / "snp_plots" / "log.txt" + LOGDIR / "af_trajectory_panel_plot" / "log.txt" script: - "../scripts/report/snp_plots.R" + "../scripts/report/af_trajectory_panel_plot.R" + + +# rule snp_plots: +# conda: "../envs/renv.yaml" +# params: +# design = config["PLOTS"], +# cor_method = config["COR"]["METHOD"], +# cor_exact = config["COR"]["EXACT"] +# input: +# vcf = OUTDIR/f"{OUTPUT_NAME}.variants.tsv", +# metadata = config["METADATA"] +# output: +# pseudovolcano = report(REPORT_DIR_PLOTS/"figure_6.png"), +# snp_panel = report(REPORT_DIR_PLOTS/"figure_7.png"), +# table_1 = report(REPORT_DIR_TABLES/"figure_6.csv"), +# table_2 = report(REPORT_DIR_TABLES/"figure_7.csv") +# log: +# LOGDIR / "snp_plots" / "log.txt" +# script: +# "../scripts/report/snp_plots.R" rule summary_table: @@ -284,8 +344,8 @@ rule report: fig_cor = report(REPORT_DIR_PLOTS/"figure_4.png"), SNV = report(REPORT_DIR_PLOTS/"figure_5a.png"), SNV_spike = report(REPORT_DIR_PLOTS/"figure_5b.png"), - volcano = report(REPORT_DIR_PLOTS/"figure_6.png"), - panel = report(REPORT_DIR_PLOTS/"figure_7.png"), + volcano = report(REPORT_DIR_TABLES/"af_time_correlation_per_snp.png"), + panel = report(REPORT_DIR_TABLES/"af_trajectory_panel.png"), tree = report(REPORT_DIR_PLOTS/"allele_freq_tree.png"), temest = report(REPORT_DIR_PLOTS/"time_signal.png"), heat_table = report(REPORT_DIR_TABLES/"heatmap.csv"), diff --git a/workflow/scripts/report/af_time_correlation_per_snp_data.R b/workflow/scripts/report/af_time_correlation_per_snp_data.R new file mode 100644 index 0000000..4186172 --- /dev/null +++ b/workflow/scripts/report/af_time_correlation_per_snp_data.R @@ -0,0 +1,111 @@ +#!/usr/bin/env Rscript + +# Write stdout and stderr to log file +log <- file(snakemake@log[[1]], open = "wt") +sink(log, type = "message") +sink(log, type = "output") + +library(tidyverse) +library(logger) +log_threshold(INFO) + +# Read data +log_info("Reading variants table") +variants <- read_delim( + snakemake@input[["variants"]], + col_select = c( + "VARIANT_NAME", + "SAMPLE", + "REGION", + "ALT_FREQ", + "POS" + ) +) %>% + # Fill positions without alt frequency with 0 + complete( + nesting(REGION, VARIANT_NAME, POS), + SAMPLE, + fill = list(ALT_FREQ = 0) + ) + +log_info("Reading metadata") +metadata <- read_csv(snakemake@input[["metadata"]]) %>% + filter( + ID %in% variants$SAMPLE + ) %>% + select( + ID, + CollectionDate + ) + +log_info("Dating variants") +variants <- left_join(variants, metadata, by = c("SAMPLE" = "ID")) %>% + # Calculate days since first sample + arrange( + CollectionDate + ) %>% + mutate( + interval = as.numeric(CollectionDate - min(CollectionDate)) + ) + +# Save processed input +log_info("Writing dated and frequency-filled variants") +write_csv(variants, snakemake@output$fmt_variants) + +log_info("Calculating correlations") +log_debug("Calculating unique SNPs") +# Get list with all different polymorphisms +unique.snps <- pull( + variants, + VARIANT_NAME +) %>% + unique() + +# Create an empty dataframe to be filled +cor.df <- data.frame( + variant = "", + coefficient = 0, + p.value = 0, + p.value.adj = 0 +) %>% + filter(p.value != 0) + +log_debug("Calculating correlation using method = {snakemake@params$cor_method} and exact p-value = {snakemake@params$cor_exact}") +correlations <- lapply( + unique.snps, + function(snp) { + # Select SNP + df <- filter( + variants, + VARIANT_NAME == snp + ) + # Perform calculation + test <- cor.test( + df$ALT_FREQ, + df$interval, + method = snakemake@params$cor_method, + exact = snakemake@params$cor_exact + ) + # Adjust p-value + p.value.adj <- p.adjust( + test$p.value, + method = "BH", + n = length(unique.snps) + ) + # Add row to dataframe + add_row( + cor.df, + variant = snp, + coefficient = test$estimate, + p.value = test$p.value, + p.value.adj = p.value.adj + ) + } +) %>% + bind_rows() + +log_info("Writing correlations table") +write_csv( + correlations, + snakemake@output[["correlations"]] +) diff --git a/workflow/scripts/report/af_time_correlation_per_snp_plot.R b/workflow/scripts/report/af_time_correlation_per_snp_plot.R new file mode 100644 index 0000000..d382cf9 --- /dev/null +++ b/workflow/scripts/report/af_time_correlation_per_snp_plot.R @@ -0,0 +1,50 @@ +#!/usr/bin/env Rscript + +# Write stdout and stderr to log file +log <- file(snakemake@log[[1]], open = "wt") +sink(log, type = "message") +sink(log, type = "output") + +library(tidyverse) +library(ggrepel) +library(logger) +log_threshold(INFO) + +# Import file with plots style +source(snakemake@params[["design"]]) + +log_info("Reading correlation data") +correlations <- read_csv(snakemake@input$correlations) + +log_info("Plotting coorrelation coefficients and p-values of each variants") +p <- correlations %>% + mutate( + trans.p = -log10(p.value.adj), + label = ifelse(p.value.adj < 0.05, variant, NA) + ) %>% + ggplot() + + aes( + x = coefficient, + y = trans.p + ) + + geom_point() + + geom_text_repel(aes(label = label), max.overlaps = 1000, direction = "x") + + xlim(c(-1, 1)) + + geom_hline( + aes(yintercept = -log10(0.05)), + linetype = 2, + color = "orange" + ) + + labs( + x = "Correlation coefficient", + y = "-log10(p-value)" + ) + +ggsave( + filename = snakemake@output$plot, + plot = p, + width = 159.2, + height = 119.4, + units = "mm", + dpi = 250 +) From c87c87f4b14053eacfa933a9147d22f0178b6369 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Fri, 17 Oct 2025 13:23:27 +0200 Subject: [PATCH 089/138] Decouple SNP AF trajectory panel plot from data Updates color selection so discard low contrast colors --- config/design_plots.R | 6 ++ workflow/rules/report.smk | 66 +++++------------- ..._snp_data.R => af_time_correlation_data.R} | 39 +++++++++-- ..._snp_plot.R => af_time_correlation_plot.R} | 2 +- .../scripts/report/af_trajectory_panel_plot.R | 68 +++++++++++++++++++ 5 files changed, 125 insertions(+), 56 deletions(-) rename workflow/scripts/report/{af_time_correlation_per_snp_data.R => af_time_correlation_data.R} (70%) rename workflow/scripts/report/{af_time_correlation_per_snp_plot.R => af_time_correlation_plot.R} (98%) create mode 100644 workflow/scripts/report/af_trajectory_panel_plot.R diff --git a/config/design_plots.R b/config/design_plots.R index 86460f3..1a07ffe 100644 --- a/config/design_plots.R +++ b/config/design_plots.R @@ -101,3 +101,9 @@ DNDS_SHAPES <- c( dN = 2, dS = 4 ) + +# Allele frequency trajectories panel color +ALL.COLORS <- grDevices::colors() +TRAJECTORY.PANEL.COLORS <- ALL.COLORS[ + !grepl("(gray|grey|white|snow|azure|beige)", ALL.COLORS) +] diff --git a/workflow/rules/report.smk b/workflow/rules/report.smk index 3a92726..fd79848 100644 --- a/workflow/rules/report.smk +++ b/workflow/rules/report.smk @@ -240,7 +240,7 @@ rule dnds_plots: "../scripts/report/dnds_plots.R" -rule af_time_correlation_per_snp_data: +rule af_time_correlation_data: conda: "../envs/renv.yaml" params: cor_method = config["COR"]["METHOD"], @@ -250,76 +250,44 @@ rule af_time_correlation_per_snp_data: metadata = config["METADATA"], output: fmt_variants = temp(OUTDIR/f"{OUTPUT_NAME}.variants.filled.dated.tsv"), - correlations = report(REPORT_DIR_TABLES/"af_time_correlation_per_snp.csv"), # fig 6 + correlations = report(REPORT_DIR_TABLES/"af_time_correlation.csv"), + subset = REPORT_DIR_TABLES/"af_time_correlation.subset.txt", log: - LOGDIR / "af_time_correlation_per_snp_data" / "log.txt" + LOGDIR / "af_time_correlation_data" / "log.txt" script: - "../scripts/report/af_time_correlation_per_snp_data.R" + "../scripts/report/af_time_correlation_data.R" -rule af_time_correlation_per_snp_plot: +rule af_time_correlation_plot: conda: "../envs/renv.yaml" params: design = config["PLOTS"], input: - correlations = REPORT_DIR_TABLES/"af_time_correlation_per_snp.csv", + correlations = REPORT_DIR_TABLES/"af_time_correlation.csv", output: - plot = report(REPORT_DIR_PLOTS/"af_time_correlation_per_snp.png"), # fig 6 + plot = report(REPORT_DIR_PLOTS/"af_time_correlation.png"), log: - LOGDIR / "af_time_correlation_per_snp_plot" / "log.txt" + LOGDIR / "af_time_correlation_plot" / "log.txt" script: - "../scripts/report/af_time_correlation_per_snp_plot.R" + "../scripts/report/af_time_correlation_plot.R" -rule af_trajectory_panel_data: # panel with AF trajectories in time - # TODO - conda: "../envs/renv.yaml" - input: - fmt_variants = OUTDIR/f"{OUTPUT_NAME}.variants.filled.dated.tsv", - correlations = REPORT_DIR_TABLES/"af_time_correlation_per_snp.csv", - output: - table = report(REPORT_DIR_TABLES/"af_trajectory_panel.csv"), # fig 7 - log: - LOGDIR / "af_trajectory_panel_data" / "log.txt" - script: - "../scripts/report/af_trajectory_panel_data.R" - - -rule af_trajectory_panel_plot: # panel with AF trajectories in time - # TODO +rule af_trajectory_panel_plot: conda: "../envs/renv.yaml" params: design = config["PLOTS"], + random_color_seed = 7291, input: - table = REPORT_DIR_TABLES/"af_trajectory_panel.csv", + fmt_variants = OUTDIR/f"{OUTPUT_NAME}.variants.filled.dated.tsv", + subset = REPORT_DIR_TABLES/"af_time_correlation.subset.txt" output: - plot = report(REPORT_DIR_PLOTS/"af_trajectory_panel.png"), # fig 7 + plot = report(REPORT_DIR_PLOTS/"af_trajectory_panel.png"), log: LOGDIR / "af_trajectory_panel_plot" / "log.txt" script: "../scripts/report/af_trajectory_panel_plot.R" -# rule snp_plots: -# conda: "../envs/renv.yaml" -# params: -# design = config["PLOTS"], -# cor_method = config["COR"]["METHOD"], -# cor_exact = config["COR"]["EXACT"] -# input: -# vcf = OUTDIR/f"{OUTPUT_NAME}.variants.tsv", -# metadata = config["METADATA"] -# output: -# pseudovolcano = report(REPORT_DIR_PLOTS/"figure_6.png"), -# snp_panel = report(REPORT_DIR_PLOTS/"figure_7.png"), -# table_1 = report(REPORT_DIR_TABLES/"figure_6.csv"), -# table_2 = report(REPORT_DIR_TABLES/"figure_7.csv") -# log: -# LOGDIR / "snp_plots" / "log.txt" -# script: -# "../scripts/report/snp_plots.R" - - rule summary_table: conda: "../envs/renv.yaml" input: @@ -344,8 +312,8 @@ rule report: fig_cor = report(REPORT_DIR_PLOTS/"figure_4.png"), SNV = report(REPORT_DIR_PLOTS/"figure_5a.png"), SNV_spike = report(REPORT_DIR_PLOTS/"figure_5b.png"), - volcano = report(REPORT_DIR_TABLES/"af_time_correlation_per_snp.png"), - panel = report(REPORT_DIR_TABLES/"af_trajectory_panel.png"), + volcano = report(REPORT_DIR_PLOTS/"af_time_correlation.png"), + panel = report(REPORT_DIR_PLOTS/"af_trajectory_panel.png"), tree = report(REPORT_DIR_PLOTS/"allele_freq_tree.png"), temest = report(REPORT_DIR_PLOTS/"time_signal.png"), heat_table = report(REPORT_DIR_TABLES/"heatmap.csv"), diff --git a/workflow/scripts/report/af_time_correlation_per_snp_data.R b/workflow/scripts/report/af_time_correlation_data.R similarity index 70% rename from workflow/scripts/report/af_time_correlation_per_snp_data.R rename to workflow/scripts/report/af_time_correlation_data.R index 4186172..6b6aafc 100644 --- a/workflow/scripts/report/af_time_correlation_per_snp_data.R +++ b/workflow/scripts/report/af_time_correlation_data.R @@ -45,7 +45,7 @@ variants <- left_join(variants, metadata, by = c("SAMPLE" = "ID")) %>% CollectionDate ) %>% mutate( - interval = as.numeric(CollectionDate - min(CollectionDate)) + interval = as.numeric(difftime(CollectionDate, min(CollectionDate), units = "days")) ) # Save processed input @@ -55,11 +55,7 @@ write_csv(variants, snakemake@output$fmt_variants) log_info("Calculating correlations") log_debug("Calculating unique SNPs") # Get list with all different polymorphisms -unique.snps <- pull( - variants, - VARIANT_NAME -) %>% - unique() +unique.snps <- unique(variants$VARIANT_NAME) # Create an empty dataframe to be filled cor.df <- data.frame( @@ -109,3 +105,34 @@ write_csv( correlations, snakemake@output[["correlations"]] ) + +log_info("Selecting variants whose allele frequency is significantly correlated with time") +significant.variants <- correlations %>% + filter(p.value.adj < 0.05) %>% + pull(variant) %>% + unique() + +log_info("Significant: {significant.variants}") + +log_info("Selecting variants in positions with more than one alternative allele") +mult.alt.variants <- variants %>% + select( + VARIANT_NAME, + POS + ) %>% + distinct() %>% + group_by(POS) %>% + filter(n() > 1) %>% + ungroup() %>% + pull(VARIANT_NAME) %>% + unique() + +log_info("Mult all: {mult.alt.variants}") + +# Build selected subset to represent +variant.selection <- unique(c(significant.variants, mult.alt.variants)) + +log_info("Selection: {variant.selection}") + +log_info("Writing selected variants subset") +write_lines(variant.selection, snakemake@output$subset) diff --git a/workflow/scripts/report/af_time_correlation_per_snp_plot.R b/workflow/scripts/report/af_time_correlation_plot.R similarity index 98% rename from workflow/scripts/report/af_time_correlation_per_snp_plot.R rename to workflow/scripts/report/af_time_correlation_plot.R index d382cf9..87321cd 100644 --- a/workflow/scripts/report/af_time_correlation_per_snp_plot.R +++ b/workflow/scripts/report/af_time_correlation_plot.R @@ -33,7 +33,7 @@ p <- correlations %>% geom_hline( aes(yintercept = -log10(0.05)), linetype = 2, - color = "orange" + color = "red" ) + labs( x = "Correlation coefficient", diff --git a/workflow/scripts/report/af_trajectory_panel_plot.R b/workflow/scripts/report/af_trajectory_panel_plot.R new file mode 100644 index 0000000..1fa71eb --- /dev/null +++ b/workflow/scripts/report/af_trajectory_panel_plot.R @@ -0,0 +1,68 @@ +#!/usr/bin/env Rscript + +# Write stdout and stderr to log file +log <- file(snakemake@log[[1]], open = "wt") +sink(log, type = "message") +sink(log, type = "output") + +set.seed(snakemake@params$random_color_seed) # seed for sampling colors + +library(tidyverse) +library(logger) +log_threshold(INFO) + +# Import file with plots style +source(snakemake@params[["design"]]) + +log_info("Reading formatted variants table") +variants <- read_csv(snakemake@input$fmt_variants) + +log_info("Reading subset of variants to represent") +selected.variants <- read_lines(snakemake@input$subset) + +# Set plot height depending on the number of SNPs assuming 4 columns in the plot +log_debug("Calculating plot height") +plot.height <- ceiling(length(selected.variants) / 4) * 42 + +log_info("Plotting {length(selected.variants)} SNPs allele frequency trajectories in time") +selected.colors <- sample(TRAJECTORY.PANEL.COLORS, length(selected.variants)) +log_debug("Selected color: {selected.colors}") +p <- variants %>% + filter(VARIANT_NAME %in% selected.variants) %>% + ggplot() + + aes( + x = interval, + y = ALT_FREQ, + color = VARIANT_NAME + ) + + scale_color_manual(values = selected.colors) + + geom_point() + + geom_line() + + theme( + legend.position = "bottom", + legend.text = element_text(size = 9) + ) + + labs( + x = "Days since first sample", + y = "Frequency", + color = "NV" + ) + + guides(color = guide_legend(ncol = 3)) + +if (length(selected.variants) > 1) { + p <- p + + facet_wrap( + vars(POS), + nrow = ceiling(length(selected.variants) / 4), + ncol = 4 + ) +} + +ggsave( + filename = snakemake@output[["plot"]], + plot = p, + width = 159.2, + height = max(100, plot.height), + units = "mm", + dpi = 250 +) From 8a70e76e218bcffaac43f2c2e673b86cb08b6ee4 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Mon, 20 Oct 2025 10:56:55 +0200 Subject: [PATCH 090/138] Remove unused script --- workflow/scripts/report/snp_plots.R | 250 ---------------------------- 1 file changed, 250 deletions(-) delete mode 100644 workflow/scripts/report/snp_plots.R diff --git a/workflow/scripts/report/snp_plots.R b/workflow/scripts/report/snp_plots.R deleted file mode 100644 index 34a95ee..0000000 --- a/workflow/scripts/report/snp_plots.R +++ /dev/null @@ -1,250 +0,0 @@ -#!/usr/bin/env Rscript - -# Write stdout and stderr to log file -log <- file(snakemake@log[[1]], open = "wt") -sink(log, type = "message") -sink(log, type = "output") - -library(tidyverse) -library(stringi) -library(ggrepel) -library(logger) -log_threshold(INFO) - -# Get colors -color <- grDevices::colors()[grep( - "gr(a|e)y", - grDevices::colors(), - invert = TRUE -)] -color <- color[grep("white", color, invert = TRUE)] -# Import file with plots style -source(snakemake@params[["design"]]) - -# DATA PREPROCESSING ##### -metadata <- read_csv(snakemake@input[["metadata"]]) - -vcf <- read_delim(snakemake@input[["vcf"]]) -data <- metadata %>% - filter( - ID %in% vcf$SAMPLE - ) %>% - dplyr::select( - ID, - CollectionDate - ) - -# Obtain sample names ordered by CollectionDate -date_order <- metadata %>% - arrange(CollectionDate) %>% - pull(ID) %>% - unique() - -# Simplify features names and create SNP variable -vcf <- vcf %>% - dplyr::select( - VARIANT_NAME, - SAMPLE, - REGION, - ALT_FREQ, - POS - ) - -# Fill positions without alt frequency with 0 -vcf <- vcf %>% - complete( - nesting(REGION, VARIANT_NAME, POS), - SAMPLE, - fill = list(ALT_FREQ = 0) - ) - -# Join variants file and metadata file -vcf <- left_join(vcf, data, by = c("SAMPLE" = "ID")) - -# Calculate days since first sample -vcf <- arrange( - vcf, - CollectionDate -) %>% - mutate( - interval = as.numeric(CollectionDate - min(CollectionDate)) - ) - -# PLOTS #### -## VOLCANO PLOT #### - -# Get list with all different polymorphisms -SNPs <- pull( - vcf, - VARIANT_NAME -) %>% - unique() - -# Create an empty dataframe to be filled -cor.df <- data.frame( - snp = "", - cor = 0, - p.value = 0 -) %>% - filter(p.value != 0) - -cor.df.fill <- lapply( - SNPs, - function(snp) { - message(snp) - df <- filter( - vcf, - VARIANT_NAME == snp - ) - - test <- cor.test( - df$ALT_FREQ, - df$interval, - method = snakemake@params$cor_method, - exact = snakemake@params$cor_exact - ) - - pvalue <- p.adjust( - test$p.value, - method = "BH", - n = length(SNPs) - ) - add_row( - cor.df, - snp = snp, - cor = test$estimate, - p.value = pvalue - ) - } -) %>% - bind_rows() - -# Plot a pseudo volcano plot with coorrelation index and p-value -log_info("Plotting pseudovolcano figure") -volcano <- cor.df.fill %>% - mutate( - trans.p = -log10(p.value), - label = case_when(p.value < 0.05 ~ snp) - ) %>% - ggplot() + - aes( - x = cor, - y = trans.p - ) + - geom_point() + - geom_text_repel(aes(label = label), max.overlaps = 1000, direction = "x") + - xlim(c(-1, 1)) + - geom_hline( - aes(yintercept = -log10(0.05)), - linetype = 2, - color = "red" - ) + - labs( - x = "Correlation", - y = "-log10(p-value)" - ) - -ggsave( - filename = snakemake@output[["pseudovolcano"]], - plot = volcano, - width = 159.2, - height = 119.4, - units = "mm", - dpi = 250 -) - - -## SNP PANEL #### -# SNPs significantly correlated with time -sign <- filter( - cor.df.fill, - p.value < 0.05 -) %>% - pull(snp) %>% - unique() - -# SNPs which are in positions with more than one alternative allele -dup <- vcf %>% - select( - VARIANT_NAME, - POS - ) %>% - unique() %>% - group_by(POS) %>% - filter(n() > 1) %>% - ungroup() %>% - pull(VARIANT_NAME) %>% - unique() - -subset <- c(sign, dup) %>% - unique() - -# Plot height depending on the number of SNPs assuming 4 columns in the plot -plot.height <- ceiling(length(subset) / 4) * 42 - -log_info("Plotting SNPs trends in time") -panel <- vcf %>% - filter(VARIANT_NAME %in% subset) %>% - ggplot() + - aes( - x = interval, - y = ALT_FREQ, - color = VARIANT_NAME - ) + - scale_color_manual(values = sample(color, length(subset))) + - geom_point() + - geom_line() + - theme( - legend.position = "bottom", - legend.text = element_text(size = 9) - ) + - labs( - x = "Days since first sample", - y = "Frequency", - color = "NV" - ) + - guides(color = guide_legend(ncol = 3)) - -if (length(subset) > 1) { - panel <- panel + - facet_wrap( - vars(POS), - nrow = ceiling(length(subset) / 4), - ncol = 4 - ) -} - -ggsave( - filename = snakemake@output[["snp_panel"]], - plot = panel, - width = 159.2, - height = max(100, plot.height), - units = "mm", - dpi = 250 -) - - -# PLOT TABLES #### -log_info("Saving coorelation table") -cor.df.fill %>% - transmute( - NV = snp, - correlation_coef = cor, - adj_pvalue = p.value - ) %>% - write.csv( - snakemake@output[["table_1"]], - row.names = FALSE - ) - -log_info("Saving SNPs trends table") -vcf %>% - filter(VARIANT_NAME %in% subset) %>% - transmute( - sample = SAMPLE, - POS = POS, - NV = VARIANT_NAME, - ALT_FREQ = ALT_FREQ, - DaysSinceFirst = interval - ) %>% - write.csv(snakemake@output[["table_2"]], row.names = FALSE) From 34c4b9c3da5768e322d3cacc9cb4546629a68a4f Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Mon, 20 Oct 2025 10:57:26 +0200 Subject: [PATCH 091/138] Set RNG seed for diversity sampling --- workflow/rules/report.smk | 1 + workflow/scripts/report/diversity_data.R | 2 ++ 2 files changed, 3 insertions(+) diff --git a/workflow/rules/report.smk b/workflow/rules/report.smk index fd79848..73fbde5 100644 --- a/workflow/rules/report.smk +++ b/workflow/rules/report.smk @@ -64,6 +64,7 @@ rule diversity_data: params: bootstrap_reps = config["DIVERSITY_REPS"], aln_reference = config["ALIGNMENT_REFERENCE"], + seed = 7291, input: study_fasta = OUTDIR/"nextalign"/f"{OUTPUT_NAME}.aligned.masked.fasta", context_fasta = OUTDIR/"context"/"nextalign"/"context_sequences.aligned.masked.fasta", diff --git a/workflow/scripts/report/diversity_data.R b/workflow/scripts/report/diversity_data.R index d852e8d..d321bde 100644 --- a/workflow/scripts/report/diversity_data.R +++ b/workflow/scripts/report/diversity_data.R @@ -5,6 +5,8 @@ log <- file(snakemake@log[[1]], open = "wt") sink(log, type = "message") sink(log, type = "output") +set.seed(snakemake@params$seed) + library(ape) library(pegas) library(future.apply) From 55fb139e341898d8e7bc817e7719acd08dae18ef Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Mon, 20 Oct 2025 12:22:44 +0200 Subject: [PATCH 092/138] Update AF trajectory panel and legend labels --- workflow/rules/report.smk | 3 +++ .../scripts/report/af_trajectory_panel_plot.R | 26 +++++++++++-------- 2 files changed, 18 insertions(+), 11 deletions(-) diff --git a/workflow/rules/report.smk b/workflow/rules/report.smk index 73fbde5..76314ba 100644 --- a/workflow/rules/report.smk +++ b/workflow/rules/report.smk @@ -277,6 +277,9 @@ rule af_trajectory_panel_plot: conda: "../envs/renv.yaml" params: design = config["PLOTS"], + n_plot_columns = 3, + plot_row_height_mm = 35, + plot_width_mm = 159.2, random_color_seed = 7291, input: fmt_variants = OUTDIR/f"{OUTPUT_NAME}.variants.filled.dated.tsv", diff --git a/workflow/scripts/report/af_trajectory_panel_plot.R b/workflow/scripts/report/af_trajectory_panel_plot.R index 1fa71eb..0094068 100644 --- a/workflow/scripts/report/af_trajectory_panel_plot.R +++ b/workflow/scripts/report/af_trajectory_panel_plot.R @@ -21,48 +21,52 @@ log_info("Reading subset of variants to represent") selected.variants <- read_lines(snakemake@input$subset) # Set plot height depending on the number of SNPs assuming 4 columns in the plot -log_debug("Calculating plot height") -plot.height <- ceiling(length(selected.variants) / 4) * 42 +plot.rows <- ceiling( + length(selected.variants) / snakemake@params$n_plot_columns +) +plot.height <- max(100, plot.rows * snakemake@params$plot_row_height_mm) +log_debug("Setting total plot height to {plot.height} mm with {plot.rows} rows") log_info("Plotting {length(selected.variants)} SNPs allele frequency trajectories in time") selected.colors <- sample(TRAJECTORY.PANEL.COLORS, length(selected.variants)) log_debug("Selected color: {selected.colors}") p <- variants %>% filter(VARIANT_NAME %in% selected.variants) %>% + mutate(gPOS = reorder(paste0("g.", POS), POS)) %>% ggplot() + aes( x = interval, y = ALT_FREQ, - color = VARIANT_NAME + color = reorder(glue::glue("{gPOS}\n{VARIANT_NAME}"), POS) ) + scale_color_manual(values = selected.colors) + geom_point() + geom_line() + theme( legend.position = "bottom", - legend.text = element_text(size = 9) + legend.text = element_text(size = 9), + legend.title = element_blank(), + legend.spacing.y = unit(3, "mm") ) + labs( x = "Days since first sample", - y = "Frequency", - color = "NV" + y = "Frequency" ) + guides(color = guide_legend(ncol = 3)) if (length(selected.variants) > 1) { p <- p + facet_wrap( - vars(POS), - nrow = ceiling(length(selected.variants) / 4), - ncol = 4 + vars(gPOS), + ncol = snakemake@params$n_plot_columns ) } ggsave( filename = snakemake@output[["plot"]], plot = p, - width = 159.2, - height = max(100, plot.height), + width = snakemake@params$plot_width_mm, + height = plot.height, units = "mm", dpi = 250 ) From bd15d31e135d372281d5afc04a06b5635efe7933 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Mon, 20 Oct 2025 17:19:35 +0200 Subject: [PATCH 093/138] Fix missing class on context tree plot Adds context tree bootstrap settings to config YAML --- config/README.md | 7 +- config/config.yaml | 10 +-- config/design_plots.R | 8 +-- workflow/rules/context.smk | 4 +- workflow/rules/report.smk | 10 +-- .../scripts/report/context_phylogeny_data.R | 7 ++ .../scripts/report/context_phylogeny_plot.R | 68 +++++++++++-------- 7 files changed, 65 insertions(+), 49 deletions(-) diff --git a/config/README.md b/config/README.md index 564dbd5..f5429e9 100644 --- a/config/README.md +++ b/config/README.md @@ -133,8 +133,11 @@ All of the following variables are pre-defined in [config.yaml](/config/config.y - `COORDINATES_JSON`: path of a JSON file containing genome feature coordinates for data visualization. - `GENETIC_CODE_JSON`: path of a JSON file containing a genetic code for gene translation. - `TREE_MODEL`: substitution model used by IQTREE (see [docs](http://www.iqtree.org/doc/Substitution-Models)). -- `UFBOOT_REPS`: ultrafast bootstrap replicates for IQTREE (see [UFBoot](https://doi.org/10.1093/molbev/msx281)). -- `SHALRT_REPS`: Shimodaira–Hasegawa approximate likelihood ratio test bootstrap replicates for IQTREE (see [SH-aLRT](https://doi.org/10.1093/sysbio/syq010)). +- `UFBOOT` & `SHALRT`: settings for ultrafast bootstrap (see [UFBoot](https://doi.org/10.1093/molbev/msx281)) + and Shimodaira–Hasegawa approximate likelihood ratio test bootstrap + (see [SH-aLRT](https://doi.org/10.1093/sysbio/syq010)) in IQTREE runs: + - `REPS`: number of replicates. + - `THRESHOLD`: value cutoff for visualization. - `VC`: variant calling configuration: - `MAX_DEPTH`: maximum depth at a position for `samtools mpileup` (option `-d`). - `MIN_QUALITY`: minimum base quality for `samtools mpileup` (option `-Q`). diff --git a/config/config.yaml b/config/config.yaml index 74f8bce..84ed754 100644 --- a/config/config.yaml +++ b/config/config.yaml @@ -8,10 +8,12 @@ GENETIC_CODE_JSON: "config/standard_genetic_code.json" TREE_MODEL: "GTR+F+I+G4" -UFBOOT_REPS: - 1000 -SHALRT_REPS: - 1000 +UFBOOT: + REPS: 1000 + THRESHOLD: 0.95 +SHALRT: + REPS: 1000 + THRESHOLD: 0.80 VC: MAX_DEPTH: 0 MIN_QUALITY: 0 diff --git a/config/design_plots.R b/config/design_plots.R index 1a07ffe..2eb17bd 100644 --- a/config/design_plots.R +++ b/config/design_plots.R @@ -60,13 +60,7 @@ TREE_NODE_SIZE <- c( TREE_LEGEND_NAMES <- c( tip_label = "Target samples", - boot_alrt_pass = sprintf( - "UFBoot ≥ %s%s & SH-aLRT ≥ %s%s ", - snakemake@params[["boot_th"]], - "%", - snakemake@params[["alrt_th"]], - "%" - ) + boot_alrt_pass = "UFBoot ≥ %.2f%s & SH-aLRT ≥ %.2f%s" ) # Nucleotide variants classification colors and labels diff --git a/workflow/rules/context.smk b/workflow/rules/context.smk index 623060c..3bb2355 100644 --- a/workflow/rules/context.smk +++ b/workflow/rules/context.smk @@ -76,8 +76,8 @@ rule ml_context_tree: params: seqtype = "DNA", name = OUTPUT_NAME, - ufboot = config["UFBOOT_REPS"], - alrt = config["SHALRT_REPS"], + ufboot = config["UFBOOT"]["REPS"], + alrt = config["SHALRT"]["REPS"], outgroup = config["ALIGNMENT_REFERENCE"], model = config["TREE_MODEL"] input: diff --git a/workflow/rules/report.smk b/workflow/rules/report.smk index 76314ba..9f56dea 100644 --- a/workflow/rules/report.smk +++ b/workflow/rules/report.smk @@ -128,8 +128,8 @@ rule context_phylogeny_data: params: design = config["PLOTS"], ref_name = config["ALIGNMENT_REFERENCE"], - boot_th = 95.0, - alrt_th = 80.0, + boot_th = config["UFBOOT"]["THRESHOLD"], + alrt_th = config["SHALRT"]["THRESHOLD"], input: target_fasta = OUTDIR/f"{OUTPUT_NAME}.fasta", tree = OUTDIR/f"tree_context/{OUTPUT_NAME}.treefile", @@ -148,6 +148,8 @@ rule context_phylogeny_plot: design = config["PLOTS"], plot_height_mm = 119.4, plot_width_mm = 159.2, + boot_th = config["UFBOOT"]["THRESHOLD"], + alrt_th = config["SHALRT"]["THRESHOLD"], input: tree = OUTDIR/f"tree_context/{OUTPUT_NAME}.treefile", json = REPORT_DIR_TABLES/"context_phylogeny.json", @@ -332,8 +334,8 @@ rule report: params: workflow_version = get_repo_version(BASE_PATH.as_posix(), __version__), min_ivar_freq = config["VC"]["IVAR_FREQ"], - ufboot_reps = config["UFBOOT_REPS"], - shalrt_reps = config["SHALRT_REPS"], + ufboot_reps = config["UFBOOT"]["REPS"], + shalrt_reps = config["SHALRT"]["REPS"], name = config["OUTPUT_NAME"], use_bionj = config["USE_BIONJ"], cor_method = config["COR"]["METHOD"], diff --git a/workflow/scripts/report/context_phylogeny_data.R b/workflow/scripts/report/context_phylogeny_data.R index 842942b..5e5eb12 100644 --- a/workflow/scripts/report/context_phylogeny_data.R +++ b/workflow/scripts/report/context_phylogeny_data.R @@ -16,6 +16,13 @@ log_threshold(INFO) # Import file with plots style source(snakemake@params[["design"]]) +# Format tree label for well supported nodes +TREE_LEGEND_NAMES["boot_alrt_pass"] <- sprintf( + TREE_LEGEND_NAMES["boot_alrt_pass"], + snakemake@params[["boot_th"]], "%", + snakemake@params[["alrt_th"]], "%" +) + tree_ml <- read.tree(snakemake@input[["tree"]]) %>% root( snakemake@params[["ref_name"]], diff --git a/workflow/scripts/report/context_phylogeny_plot.R b/workflow/scripts/report/context_phylogeny_plot.R index 1d519a8..dd6baf8 100644 --- a/workflow/scripts/report/context_phylogeny_plot.R +++ b/workflow/scripts/report/context_phylogeny_plot.R @@ -16,46 +16,54 @@ log_threshold(INFO) # Import file with plots style source(snakemake@params[["design"]]) +# Format tree label for well supported nodes +TREE_LEGEND_NAMES["boot_alrt_pass"] <- sprintf( + TREE_LEGEND_NAMES["boot_alrt_pass"], + snakemake@params[["boot_th"]], "%", + snakemake@params[["alrt_th"]], "%" +) + log_info("Reading JSON data") json <- read_json(snakemake@input$json) log_info("Reading tree") tree_ml <- read.tree(snakemake@input[["tree"]]) %>% - root( - json$root, - resolve.root = TRUE - ) + root( + json$root, + resolve.root = TRUE + ) log_info("Reading tree annotation") annotation <- read_csv(snakemake@input$annotation) log_info("Plotting M-L tree with context samples") -p <- ggtree(tree_ml, layout = "circular") %<+% annotation + - geom_highlight(node = json$target_mrca, colour = "red", alpha = 0) + - geom_point(aes(color = Class, size = Class)) + - geom_treescale(1.05 * json$max_tip_length) + - geom_rootedge(0.05 * json$max_tip_length) + - xlim(-json$max_tip_length / 3, NA) + - scale_color_manual( - values = setNames( - TREE_PALETTE[names(TREE_LEGEND_NAMES)], - TREE_LEGEND_NAMES - ), - na.translate = FALSE - ) + - scale_size_manual( - values = setNames( - TREE_NODE_SIZE[names(TREE_LEGEND_NAMES)], - TREE_LEGEND_NAMES - ), - na.translate = FALSE - ) +p <- ggtree(tree_ml, layout = "circular") %<+% + annotation + + geom_highlight(node = json$target_mrca, colour = "red", alpha = 0) + + geom_point(aes(color = Class, size = Class)) + + geom_treescale(1.05 * json$max_tip_length) + + geom_rootedge(0.05 * json$max_tip_length) + + xlim(-json$max_tip_length / 3, NA) + + scale_color_manual( + values = setNames( + TREE_PALETTE[names(TREE_LEGEND_NAMES)], + TREE_LEGEND_NAMES + ), + na.translate = FALSE + ) + + scale_size_manual( + values = setNames( + TREE_NODE_SIZE[names(TREE_LEGEND_NAMES)], + TREE_LEGEND_NAMES + ), + na.translate = FALSE + ) ggsave( - filename = snakemake@output[["plot"]], - plot = p, - width = snakemake@params[["plot_width_mm"]], - height = snakemake@params[["plot_height_mm"]], - units = "mm", - dpi = 250 + filename = snakemake@output[["plot"]], + plot = p, + width = snakemake@params[["plot_width_mm"]], + height = snakemake@params[["plot_height_mm"]], + units = "mm", + dpi = 250 ) From de1e170e4ea38c5622b4a573fe9d992c71021fa3 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Thu, 30 Oct 2025 16:18:54 +0100 Subject: [PATCH 094/138] Filter GenBank features in a separate rule --- workflow/rules/evolution.smk | 19 ++++- workflow/scripts/N_S_sites_from_fasta.py | 62 +-------------- workflow/scripts/filter_genbank_features.py | 83 +++++++++++++++++++++ 3 files changed, 102 insertions(+), 62 deletions(-) create mode 100644 workflow/scripts/filter_genbank_features.py diff --git a/workflow/rules/evolution.smk b/workflow/rules/evolution.smk index 6edcb97..2951763 100644 --- a/workflow/rules/evolution.smk +++ b/workflow/rules/evolution.smk @@ -1,12 +1,27 @@ +rule filter_genbank_features: + threads: 1 + conda: "../envs/biopython.yaml" + params: + included = config.get("GB_FEATURES", {}).get("INCLUDE", {}), + excluded = config.get("GB_FEATURES", {}).get("EXCLUDE", {}), + input: + gb = OUTDIR/"reference.gb", + output: + gb = OUTDIR/"reference.cds.gb", + log: + LOGDIR / "filter_genbank_features" / "log.txt" + script: + "../scripts/filter_genbank_features.py" + + rule N_S_sites: threads: 1 conda: "../envs/biopython.yaml" params: - features = config.get("GB_FEATURES", {}), gb_qualifier_display = "gene", input: fasta = OUTDIR/f"{OUTPUT_NAME}.ancestor.fasta", - gb = OUTDIR/"reference.gb", + gb = OUTDIR/"reference.cds.gb", genetic_code = Path(config["GENETIC_CODE_JSON"]).resolve(), output: csv = temp(OUTDIR/f"{OUTPUT_NAME}.ancestor.N_S.sites.csv"), diff --git a/workflow/scripts/N_S_sites_from_fasta.py b/workflow/scripts/N_S_sites_from_fasta.py index 97cd10d..cb7fa6f 100644 --- a/workflow/scripts/N_S_sites_from_fasta.py +++ b/workflow/scripts/N_S_sites_from_fasta.py @@ -3,12 +3,11 @@ import logging import json import itertools as it -from typing import Dict, Iterable +from typing import Dict import pandas as pd from Bio import SeqIO from Bio.SeqRecord import SeqRecord -from Bio.SeqFeature import SeqFeature from Bio.Seq import Seq @@ -69,54 +68,6 @@ def calculate_ns_sites(codons: dict, genetic_code: dict) -> pd.DataFrame: return pd.DataFrame({"feature": features, "N": N_sites, "S": S_sites}) -def iter_features_filtering(features: Iterable[SeqFeature], included: Dict[str, str], excluded: Dict[str, str]) -> Iterable[SeqFeature]: - # No filters - if len(included) == 0 and len(excluded) == 0: - logging.debug("Selecting all features") - return iter(features) - # Only inclusion filter - elif len(included) == 0 and len(excluded) != 0: - logging.debug(f"Selecting features excluding all of {excluded}") - return ( - feature for feature in features - if all( - (qualifier_value not in excluded.get(qualifier_key, [])) - for qualifier_key in excluded.keys() - for qualifier_value in feature.qualifiers.get(qualifier_key, []) - ) - ) - # Only exclusion filter - elif len(included) != 0 and len(excluded) == 0: - logging.debug(f"Selecting features including any of {included}") - return ( - feature for feature in features - if any( - (qualifier_value in included.get(qualifier_key, [])) - for qualifier_key in included.keys() - for qualifier_value in feature.qualifiers.get(qualifier_key, []) - ) - ) - # Inclusion then exclusion filter - else: - logging.debug(f"Selecting features including any of {included} and then excluding all of {excluded}") - included_features = ( - feature for feature in features - if any( - (qualifier_value in included.get(qualifier_key, [])) - for qualifier_key in included.keys() - for qualifier_value in feature.qualifiers.get(qualifier_key, []) - ) - ) - return ( - feature for feature in included_features - if all( - (qualifier_value not in excluded.get(qualifier_key, [])) - for qualifier_key in excluded.keys() - for qualifier_value in feature.qualifiers.get(qualifier_key, []) - ) - ) - - def main(): logging.basicConfig( @@ -136,16 +87,7 @@ def main(): logging.info("Extracting CDS") coding_records = {} - included = snakemake.params.features.get("INCLUDE", {}) - excluded = snakemake.params.features.get("EXCLUDE", {}) - for feature in iter_features_filtering(gb.features, included, excluded): - logging.debug( - "Processing SeqFeature: " - f"ID={feature.id} type={feature.type} location={feature.location} " - f"gene={feature.qualifiers.get('gene', [])} " - f"locus_tag={feature.qualifiers.get('locus_tag', [])} " - f"product={feature.qualifiers.get('product', [])}" - ) + for feature in gb.features: identifier = "|".join(feature.qualifiers.get(snakemake.params.gb_qualifier_display, [])) if identifier == "": logging.error(f"Feature at {feature.location} has no qualifier '{snakemake.params.gb_qualifier_display}'") diff --git a/workflow/scripts/filter_genbank_features.py b/workflow/scripts/filter_genbank_features.py new file mode 100644 index 0000000..959a24e --- /dev/null +++ b/workflow/scripts/filter_genbank_features.py @@ -0,0 +1,83 @@ +#!/usr/bin/env python3 + +import logging +from typing import Dict, Iterable + +from Bio import SeqIO +from Bio.SeqFeature import SeqFeature + + +def iter_features_filtering(features: Iterable[SeqFeature], included: Dict[str, str], excluded: Dict[str, str]) -> Iterable[SeqFeature]: + # No filters + if len(included) == 0 and len(excluded) == 0: + logging.debug("Selecting all features") + return iter(features) + # Only inclusion filter + elif len(included) == 0 and len(excluded) != 0: + logging.debug(f"Selecting features excluding all of {excluded}") + return ( + feature for feature in features + if all( + (qualifier_value not in excluded.get(qualifier_key, [])) + for qualifier_key in excluded.keys() + for qualifier_value in feature.qualifiers.get(qualifier_key, []) + ) + ) + # Only exclusion filter + elif len(included) != 0 and len(excluded) == 0: + logging.debug(f"Selecting features including any of {included}") + return ( + feature for feature in features + if any( + (qualifier_value in included.get(qualifier_key, [])) + for qualifier_key in included.keys() + for qualifier_value in feature.qualifiers.get(qualifier_key, []) + ) + ) + # Inclusion then exclusion filter + else: + logging.debug(f"Selecting features including any of {included} and then excluding all of {excluded}") + included_features = ( + feature for feature in features + if any( + (qualifier_value in included.get(qualifier_key, [])) + for qualifier_key in included.keys() + for qualifier_value in feature.qualifiers.get(qualifier_key, []) + ) + ) + return ( + feature for feature in included_features + if all( + (qualifier_value not in excluded.get(qualifier_key, [])) + for qualifier_key in excluded.keys() + for qualifier_value in feature.qualifiers.get(qualifier_key, []) + ) + ) + + +if __name__ == "__main__": + logging.basicConfig( + filename=snakemake.log[0], format=snakemake.config["LOG_PY_FMT"], + level=logging.INFO + ) + + logging.info("Reading GenBank file") + gb = SeqIO.read(snakemake.input.gb, format="gb") + + logging.info("Extracting CDS") + features = [] + for feature in iter_features_filtering(gb.features, snakemake.params.included, snakemake.params.excluded): + logging.debug( + "Processing SeqFeature: " + f"ID={feature.id} type={feature.type} location={feature.location} " + f"gene={feature.qualifiers.get('gene', [])} " + f"locus_tag={feature.qualifiers.get('locus_tag', [])} " + f"product={feature.qualifiers.get('product', [])}" + ) + features.append(feature) + + logging.info("Replacing features") + gb.features = features + + logging.info("Writing filtered GenBank files") + SeqIO.write(gb, snakemake.output.gb, "gb") From 4917f216ab55ef5183badbe4b02aa2aa1c0d4e67 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Thu, 30 Oct 2025 16:55:43 +0100 Subject: [PATCH 095/138] Extract NV panel regions from GenBank file Removes the need of a JSON file with coordinates, but is flexible enough to allow its usage --- .test/targets.yaml | 2 - config/README.md | 1 - config/config.yaml | 2 - config/sarscov2_coordinates.json | 23 --------- workflow/rules/report.smk | 22 +++++++-- .../scripts/report/extract_genbank_regions.py | 48 +++++++++++++++++++ 6 files changed, 66 insertions(+), 32 deletions(-) delete mode 100644 config/sarscov2_coordinates.json create mode 100644 workflow/scripts/report/extract_genbank_regions.py diff --git a/.test/targets.yaml b/.test/targets.yaml index 652a5fb..dfbcb7c 100644 --- a/.test/targets.yaml +++ b/.test/targets.yaml @@ -40,7 +40,5 @@ PLOT_GENOME_REGIONS: "config/nsp_annotation.csv" REPORT_QMD: "template.qmd" -COORDINATES_JSON: - "config/sarscov2_coordinates.json" GENETIC_CODE_JSON: "config/standard_genetic_code.json" diff --git a/config/README.md b/config/README.md index f5429e9..e902341 100644 --- a/config/README.md +++ b/config/README.md @@ -130,7 +130,6 @@ All of the following variables are pre-defined in [config.yaml](/config/config.y - `ALIGNMENT_REFERENCE`: NCBI accession number of the reference record for sequence alignment. - `PROBLEMATIC_VCF`: URL or path of a VCF file containing problematic genome positions for masking. -- `COORDINATES_JSON`: path of a JSON file containing genome feature coordinates for data visualization. - `GENETIC_CODE_JSON`: path of a JSON file containing a genetic code for gene translation. - `TREE_MODEL`: substitution model used by IQTREE (see [docs](http://www.iqtree.org/doc/Substitution-Models)). - `UFBOOT` & `SHALRT`: settings for ultrafast bootstrap (see [UFBoot](https://doi.org/10.1093/molbev/msx281)) diff --git a/config/config.yaml b/config/config.yaml index 84ed754..c00cc02 100644 --- a/config/config.yaml +++ b/config/config.yaml @@ -2,8 +2,6 @@ ALIGNMENT_REFERENCE: "NC_045512.2" PROBLEMATIC_VCF: "https://raw.githubusercontent.com/W-L/ProblematicSites_SARS-CoV2/da322c32004f7b16bdaa6a8ee7fd24d56e79d9dc/problematic_sites_sarsCov2.vcf" -COORDINATES_JSON: - "config/sarscov2_coordinates.json" GENETIC_CODE_JSON: "config/standard_genetic_code.json" TREE_MODEL: diff --git a/config/sarscov2_coordinates.json b/config/sarscov2_coordinates.json deleted file mode 100644 index a7242f5..0000000 --- a/config/sarscov2_coordinates.json +++ /dev/null @@ -1,23 +0,0 @@ -{ - "five_prime_UTR": [1, 265], - "ORF1ab": [266, 21555], - "Intergenic_1": [21556, 21562], - "S": [21563, 25384], - "Intergenic_2": [25385, 25392], - "ORF3a": [25393, 26220], - "Intergenic_3": [26221, 26244], - "E": [26245, 26472], - "Intergenic_4": [26473, 26522], - "M": [26523, 27191], - "Intergenic_5": [27192, 27201], - "ORF6": [27202, 27387], - "Intergenic_6": [27388, 27393], - "ORF7": [27394, 27759], - "Intergenic_7": [27760, 27893], - "ORF8": [27894, 28259], - "Intergenic_8": [28260, 28273], - "N": [28274, 29533], - "Intergenic_9": [29534, 29557], - "ORF10": [29558, 29674], - "three_prime_UTR": [29675, 29903] -} diff --git a/workflow/rules/report.smk b/workflow/rules/report.smk index 9f56dea..32482cb 100644 --- a/workflow/rules/report.smk +++ b/workflow/rules/report.smk @@ -95,6 +95,20 @@ rule diversity_plot: "../scripts/report/diversity_plot.R" +rule extract_genbank_regions: + conda: "../envs/biopython.yaml" + params: + gb_qualifier = "gene", + input: + gb = OUTDIR/"reference.cds.gb", + output: + regions = temp(REPORT_DIR_TABLES/"genbank_regions.json"), + log: + LOGDIR / "extract_genbank_regions" / "log.txt" + script: + "../scripts/report/extract_genbank_regions.py" + + rule general_NV_description: conda: "../envs/renv.yaml" params: @@ -104,15 +118,15 @@ rule general_NV_description: step = config["WINDOW"]["STEP"], max_alt_freq = 1.0 - config["VC"]["IVAR_FREQ"] input: - coordinates = config["COORDINATES_JSON"], + coordinates = REPORT_DIR_TABLES/"genbank_regions.json", regions = config["PLOT_GENOME_REGIONS"], window = OUTDIR/f"{OUTPUT_NAME}.window.csv", vcf = OUTDIR/f"{OUTPUT_NAME}.variants.tsv", metadata = config["METADATA"] output: - fig = report(REPORT_DIR_PLOTS/"figure_5a.png"), - fig_s = report(REPORT_DIR_PLOTS/"figure_5b.png"), - fig_cor = report(REPORT_DIR_PLOTS/"figure_4.png"), + fig = report(REPORT_DIR_PLOTS/"figure_5a.png"), # panel + fig_s = report(REPORT_DIR_PLOTS/"figure_5b.png"), # panel spike + fig_cor = report(REPORT_DIR_PLOTS/"figure_4.png"), # SNPs vs time json = temp(OUTDIR/"summary_nv.json"), table_1 = report(REPORT_DIR_TABLES/"figure_5a.csv"), table_2 = report(REPORT_DIR_TABLES/"figure_5b.csv"), diff --git a/workflow/scripts/report/extract_genbank_regions.py b/workflow/scripts/report/extract_genbank_regions.py new file mode 100644 index 0000000..61f3a33 --- /dev/null +++ b/workflow/scripts/report/extract_genbank_regions.py @@ -0,0 +1,48 @@ +#!/usr/bin/env python3 + +import logging +import json + +from Bio import SeqIO +from Bio.SeqFeature import ExactPosition + + +if __name__ == "__main__": + logging.basicConfig( + filename=snakemake.log[0], format=snakemake.config["LOG_PY_FMT"], + level=logging.INFO + ) + + logging.info("Reading GenBank file") + gb = SeqIO.read(snakemake.input.gb, format="gb") + + logging.info("Calculating CDS regions") + regions = {} + for feature in gb.features: + identifier = "|".join(feature.qualifiers.get(snakemake.params.gb_qualifier, [])) + if identifier == "": + logging.error(f"Feature at {feature.location} has no qualifier '{snakemake.params.gb_qualifier_display}'") + elif identifier in regions: + logging.warning(f"Identifier '{identifier}' is already among coding records and will not be replaced by feature at {feature.location}") + else: + logging.debug(f"Adding feature") + if type(feature.location.start) is not ExactPosition or type(feature.location.start) is not ExactPosition: + logging.warning(f"Feature '{identifier}' location is not exact but will be treated as such: {feature.location}") + regions[identifier] = ( + int(feature.location.start.real + 1), + int(feature.location.end.real) + ) + + logging.info("Calculating intergenic regions") + cds_names = tuple(regions.keys()) + intergenic_count = 0 + for i in range(1, len(cds_names)): + start1, end1 = regions[cds_names[i-1]] + start2, end2 = regions[cds_names[i]] + if (start2 - end1) > 1: + intergenic_count += 1 + regions[f"Intergenic_{intergenic_count}"] = (end1 + 1, start2 - 1) + + logging.info("Writing regions to JSON file") + with open(snakemake.output.regions, "w") as fw: + json.dump(regions, fw, indent=2) From 8daeea0d5cc3c98e7656966a58556fc9145c315b Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Fri, 31 Oct 2025 15:53:04 +0100 Subject: [PATCH 096/138] Decouple of polymorphic sites over time plot from data --- .../report/polymorphic_sites_over_time_data.R | 79 +++++++++++++++++++ .../report/polymorphic_sites_over_time_plot.R | 42 ++++++++++ 2 files changed, 121 insertions(+) create mode 100644 workflow/scripts/report/polymorphic_sites_over_time_data.R create mode 100644 workflow/scripts/report/polymorphic_sites_over_time_plot.R diff --git a/workflow/scripts/report/polymorphic_sites_over_time_data.R b/workflow/scripts/report/polymorphic_sites_over_time_data.R new file mode 100644 index 0000000..bf328f6 --- /dev/null +++ b/workflow/scripts/report/polymorphic_sites_over_time_data.R @@ -0,0 +1,79 @@ +#!/usr/bin/env Rscript + +# Write stdout and stderr to log file +log <- file(snakemake@log[[1]], open = "wt") +sink(log, type = "message") +sink(log, type = "output") + +library(tidyverse) +library(jsonlite) +library(logger) +log_threshold(INFO) + +log_info("Reading variants") +variants <- read_delim(snakemake@input$variants) + +log_info("Reading metadata") +metadata <- read_delim(snakemake@input$metadata) + +log_info("Calculating heterozygous sites") +sites <- variants %>% + filter(ALT_FREQ <= snakemake@params$max_alt_freq) %>% + left_join( + metadata, + by = c("SAMPLE" = "ID") + ) %>% + group_by(SAMPLE) %>% + summarise( + CollectionDate = min(as.Date(CollectionDate)), + n = n_distinct(POS) + ) %>% + ungroup() %>% + arrange(CollectionDate) %>% + mutate( + Day = as.numeric( + difftime(CollectionDate, min(CollectionDate), units = "days") + ) + ) + +if (nrow(sites) == 0) { + log_warn("There are none, using an empty table and no linear regression") + sites <- tibble( + SAMPLE = date_order, + REGION = as.character(NA), + VARIANT_NAME = as.character(NA), + ALT_FREQ = as.numeric(NA), + EFFECT = as.character(NA), + SYNONYMOUS = as.character(NA), + POS = as.numeric(NA), + ALT = as.character(NA), + NV_class = as.character(NA), + group = as.character(NA) + ) + r_squared <- "none" + p_value_string <- "none" +} else if (nrow(sites) > 2) { + log_info("Calculating linear regression") + model <- lm(n ~ CollectionDate, data = sites) + r_squared <- summary(model)$r.squared[[1]] + p_value <- summary(model)$coefficients[2, 4] + p_value_string <- ifelse(p_value < 0.001, "< 0.001", p_value) +} else { + log_warn("Not enough data points for a linear regression") + r_squared <- "none" + p_value_string <- "none" +} + +log_info("Writing JSON summary") +list( + "r2" = r_squared, + "value" = p_value_string +) %>% + write_json( + snakemake@output$json, + auto_unbox = TRUE, + digits = NA + ) + +log_info("Writing processed table") +write_csv(sites, snakemake@output$table) diff --git a/workflow/scripts/report/polymorphic_sites_over_time_plot.R b/workflow/scripts/report/polymorphic_sites_over_time_plot.R new file mode 100644 index 0000000..40564a3 --- /dev/null +++ b/workflow/scripts/report/polymorphic_sites_over_time_plot.R @@ -0,0 +1,42 @@ +#!/usr/bin/env Rscript + +# Write stdout and stderr to log file +log <- file(snakemake@log[[1]], open = "wt") +sink(log, type = "message") +sink(log, type = "output") + +library(tidyverse) +library(logger) +log_threshold(INFO) + +# Import file with plots style +source(snakemake@params[["design"]]) + +log_info("Reading plot data") +df <- read_csv(snakemake@input$table) + +log_info("Plotting") +p <- df %>% + ggplot() + + aes(x = Day, y = n) + + geom_smooth( + method = "lm", + fill = "gray95", + alpha = 0.6, + colour = "orange" + ) + + geom_point(size = 1, shape = 1) + + labs( + x = "Days since the initial sampling", + y = "No. of polimorphic sites" + ) + +log_info("Saving plot") +ggsave( + filename = snakemake@output$plot, + plot = p, + width = 250, + height = 119.4, + units = "mm", + dpi = 250 +) From 7978a484961e5b9edee52054835e36aa3d20c694 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Fri, 31 Oct 2025 15:54:08 +0100 Subject: [PATCH 097/138] Decouple NV panel plot from data --- config/README.md | 2 +- config/nsp_annotation.csv | 22 +- workflow/rules/fetch.smk | 5 + workflow/rules/report.smk | 139 ++++- workflow/scripts/report/NV_description.R | 488 ------------------ workflow/scripts/report/nv_panel_data.R | 104 ++++ workflow/scripts/report/nv_panel_plot.R | 160 ++++++ .../report/nv_panel_zoom_on_feature_data.py | 28 + .../report/window_zoom_on_feature_data.py | 28 + workflow/scripts/window.py | 8 + 10 files changed, 455 insertions(+), 529 deletions(-) delete mode 100644 workflow/scripts/report/NV_description.R create mode 100644 workflow/scripts/report/nv_panel_data.R create mode 100644 workflow/scripts/report/nv_panel_plot.R create mode 100644 workflow/scripts/report/nv_panel_zoom_on_feature_data.py create mode 100644 workflow/scripts/report/window_zoom_on_feature_data.py diff --git a/config/README.md b/config/README.md index e902341..04a94cb 100644 --- a/config/README.md +++ b/config/README.md @@ -180,7 +180,7 @@ All of the following variables are pre-defined in [config.yaml](/config/config.y - `EXACT`: boolean flag indicating whether to compute an exact p-value when possible. This option applies only to certain methods and may be set to `null` (default) to let R decide automatically. - `LOG_PY_FMT`: logging format string for Python scripts. - `PLOTS`: path of the R script that sets the design and style of data visualizations. -- `PLOT_GENOME_REGIONS`: path of a CSV file containing genome regions, e.g. SARS-CoV-2 non-structural protein (NSP) coordinates, for data visualization. +- `PLOT_GENOME_REGIONS`: path of a CSV file containing genome regions, e.g. SARS-CoV-2 non-structural protein (NSP) coordinates, for data visualization (columns: `region`, `start`, `end`). - `REPORT_QMD`: path of the report template in Quarto markdown (QMD) format. TODO: describe new entries in config.yaml diff --git a/config/nsp_annotation.csv b/config/nsp_annotation.csv index 02126d0..78ab294 100644 --- a/config/nsp_annotation.csv +++ b/config/nsp_annotation.csv @@ -1,17 +1,7 @@ region,start,end -nsp1,266,805 -nsp2,806,2719 -nsp3,2720,8554 -nsp4,8555,10054 -nsp5,10055,10972 -nsp6,10973,11842 -nsp7,11843,12091 -nsp8,12092,12685 -nsp9,12686,13024 -nsp10,13025,13441 -nsp11,13442,13468 -nsp12,13468,16236 -nsp13,16237,18039 -nsp14,18040,19620 -nsp15,19621,20658 -nsp16,20659,21552 +NSP2,806,2719 +NSP3,2720,8554 +NSP4-11,8555,13468 +RdRP,13468,16236 +NSP13,16237,18039 +NSP14-16,18040,21552 diff --git a/workflow/rules/fetch.smk b/workflow/rules/fetch.smk index 9f290be..3e4860c 100644 --- a/workflow/rules/fetch.smk +++ b/workflow/rules/fetch.smk @@ -66,3 +66,8 @@ rule fetch_problematic_vcf: select_problematic_vcf() shell: "curl {params.url} -o {output} -s 2> {log}" + + +rule create_empty_file: + output: + temp(touch(OUTDIR/"empty.txt")) diff --git a/workflow/rules/report.smk b/workflow/rules/report.smk index 32482cb..29dfa4b 100644 --- a/workflow/rules/report.smk +++ b/workflow/rules/report.smk @@ -51,7 +51,8 @@ rule window: variants = OUTDIR/f"{OUTPUT_NAME}.variants.tsv", gb = OUTDIR/"reference.gb", output: - window_df = temp(OUTDIR/f"{OUTPUT_NAME}.window.csv"), + window_df = temp(REPORT_DIR_TABLES/"window.csv"), + json = temp(REPORT_DIR_TABLES/"window.json"), log: LOGDIR / "window" / "log.txt" script: @@ -109,32 +110,122 @@ rule extract_genbank_regions: "../scripts/report/extract_genbank_regions.py" -rule general_NV_description: +rule polymorphic_sites_over_time_data: + conda: "../envs/renv.yaml" + params: + max_alt_freq = 1.0 - config["VC"]["IVAR_FREQ"], + input: + variants = OUTDIR/f"{OUTPUT_NAME}.variants.tsv", + metadata = config["METADATA"], + output: + table = REPORT_DIR_PLOTS/"polymorphic_sites_over_time.csv", + json = temp(REPORT_DIR_TABLES/"polymorphic_sites_over_time.json"), + log: + LOGDIR / "polymorphic_sites_over_time_data" / "log.txt" + script: + "../scripts/report/polymorphic_sites_over_time_data.R" + + +rule polymorphic_sites_over_time_plot: conda: "../envs/renv.yaml" params: - samples = expand("{sample}", sample = iter_samples()), design = config["PLOTS"], - window = config["WINDOW"]["WIDTH"], - step = config["WINDOW"]["STEP"], - max_alt_freq = 1.0 - config["VC"]["IVAR_FREQ"] input: - coordinates = REPORT_DIR_TABLES/"genbank_regions.json", - regions = config["PLOT_GENOME_REGIONS"], - window = OUTDIR/f"{OUTPUT_NAME}.window.csv", - vcf = OUTDIR/f"{OUTPUT_NAME}.variants.tsv", - metadata = config["METADATA"] + table = REPORT_DIR_PLOTS/"polymorphic_sites_over_time.csv", output: - fig = report(REPORT_DIR_PLOTS/"figure_5a.png"), # panel - fig_s = report(REPORT_DIR_PLOTS/"figure_5b.png"), # panel spike - fig_cor = report(REPORT_DIR_PLOTS/"figure_4.png"), # SNPs vs time - json = temp(OUTDIR/"summary_nv.json"), - table_1 = report(REPORT_DIR_TABLES/"figure_5a.csv"), - table_2 = report(REPORT_DIR_TABLES/"figure_5b.csv"), - table_3 = report(REPORT_DIR_TABLES/"figure_4.csv") + plot = report(REPORT_DIR_PLOTS/"polymorphic_sites_over_time.png"), log: - LOGDIR / "general_NV_description" / "log.txt" + LOGDIR / "polymorphic_sites_over_time_plot" / "log.txt" script: - "../scripts/report/NV_description.R" + "../scripts/report/polymorphic_sites_over_time_plot.R" + + +rule nv_panel_data: + conda: "../envs/renv.yaml" + input: + variants = OUTDIR/f"{OUTPUT_NAME}.variants.tsv", + metadata = config["METADATA"], + output: + table = REPORT_DIR_TABLES/"nv_panel.csv", + json = temp(REPORT_DIR_TABLES/"nv_panel.json"), + log: + LOGDIR / "nv_panel_data" / "log.txt" + script: + "../scripts/report/nv_panel_data.R" + + +rule nv_panel_zoom_on_feature_data: + input: + table = REPORT_DIR_TABLES/"nv_panel.csv", + regions = REPORT_DIR_TABLES/"genbank_regions.json", + output: + table = temp(REPORT_DIR_TABLES/"nv_panel.{region_name}.csv"), + log: + LOGDIR / "nv_panel_zoom_on_feature_data" / "{region_name}.log.txt" + script: + "../scripts/report/nv_panel_zoom_on_feature_data.py" + + +rule window_zoom_on_feature_data: + input: + table = REPORT_DIR_TABLES/"window.csv", + regions = REPORT_DIR_TABLES/"genbank_regions.json", + output: + table = temp(REPORT_DIR_TABLES/"window.{region_name}.csv"), + log: + LOGDIR / "window_zoom_on_feature_data" / "{region_name}.log.txt" + script: + "../scripts/report/window_zoom_on_feature_data.py" + + +rule nv_panel_plot: + conda: "../envs/renv.yaml" + params: + design = config["PLOTS"], + window_step = config["WINDOW"]["STEP"], + plot_height_mm = 250, + plot_width_mm = 240, + input: + panel = REPORT_DIR_TABLES/"nv_panel.csv", + window = REPORT_DIR_TABLES/"window.csv", + regions = REPORT_DIR_TABLES/"genbank_regions.json", + highlight_window_regions = config["PLOT_GENOME_REGIONS"], + output: + plot = report(REPORT_DIR_PLOTS/"nv_panel.png"), + log: + LOGDIR / "nv_panel_plot" / "log.txt" + script: + "../scripts/report/nv_panel_plot.R" + + +use rule nv_panel_plot as nv_panel_plot_S with: + input: + panel = REPORT_DIR_TABLES/"nv_panel.S.csv", + window = REPORT_DIR_TABLES/"window.S.csv", + regions = REPORT_DIR_TABLES/"genbank_regions.json", + highlight_window_regions = OUTDIR/"empty.txt", + output: + plot = report(REPORT_DIR_PLOTS/"nv_panel.S.png"), + log: + LOGDIR / "nv_panel_plot_S" / "log.txt" + + +rule merge_json_files: + input: + REPORT_DIR_TABLES/"nv_panel.json", + REPORT_DIR_TABLES/"polymorphic_sites_over_time.json", + REPORT_DIR_TABLES/"window.json", + output: + json = REPORT_DIR_TABLES/"nv_panel_summary.json", + run: + import json + result = {} + for path in input: + with open(path) as f: + d = json.load(f) + result |= d # will replace existing keys + with open(output.json, "w") as fw: + json.dump(result, fw, indent=2) rule context_phylogeny_data: @@ -329,9 +420,9 @@ rule report: demix = report(REPORT_DIR_PLOTS/"demix.png"), tree_ml = report(REPORT_DIR_PLOTS/"context_phylogeny.png"), diversity = report(REPORT_DIR_PLOTS/"diversity.png"), - fig_cor = report(REPORT_DIR_PLOTS/"figure_4.png"), - SNV = report(REPORT_DIR_PLOTS/"figure_5a.png"), - SNV_spike = report(REPORT_DIR_PLOTS/"figure_5b.png"), + fig_cor = report(REPORT_DIR_PLOTS/"polymorphic_sites_over_time.png"), + SNV = report(REPORT_DIR_PLOTS/"nv_panel.png"), + SNV_spike = report(REPORT_DIR_PLOTS/"nv_panel.S.png"), volcano = report(REPORT_DIR_PLOTS/"af_time_correlation.png"), panel = report(REPORT_DIR_PLOTS/"af_trajectory_panel.png"), tree = report(REPORT_DIR_PLOTS/"allele_freq_tree.png"), @@ -344,7 +435,7 @@ rule report: stats_lm = REPORT_DIR_TABLES/"time_signal.json", stats_ml = REPORT_DIR_TABLES/"context_phylogeny.json", table = OUTDIR/"summary_table.csv", - sum_nv = OUTDIR/"summary_nv.json", + sum_nv = REPORT_DIR_TABLES/"nv_panel_summary.json", params: workflow_version = get_repo_version(BASE_PATH.as_posix(), __version__), min_ivar_freq = config["VC"]["IVAR_FREQ"], diff --git a/workflow/scripts/report/NV_description.R b/workflow/scripts/report/NV_description.R deleted file mode 100644 index 5e9ffd1..0000000 --- a/workflow/scripts/report/NV_description.R +++ /dev/null @@ -1,488 +0,0 @@ -#!/usr/bin/env Rscript - -# Write stdout and stderr to log file -log <- file(snakemake@log[[1]], open = "wt") -sink(log, type = "message") -sink(log, type = "output") - -library(tidyverse) -library(stringi) -library(ggpubr) -library(jsonlite) -library(logger) -log_threshold(INFO) - - -# Import file with plots style -source(snakemake@params[["design"]]) - -log_info("Reading inputs") - -# Anotation for genome scheme -coordinates <- lapply( - read_json(snakemake@input$coordinates, simplifyVector = TRUE), - function(x) x[1]:x[2] -) - -vcf <- read_delim(snakemake@input[["vcf"]]) -vcf_snp <- vcf -window <- read_csv(snakemake@input[["window"]]) -metadata <- read_csv(snakemake@input[["metadata"]]) - -# DATA PROCESSING -# Obtain sample names ordered by CollectionDate -date_order <- metadata %>% - filter(ID %in% snakemake@params[["samples"]]) %>% - arrange(CollectionDate) %>% - pull(ID) %>% - unique() - -empty_vcf <- tibble( - SAMPLE = date_order, - REGION = as.character(NA), - VARIANT_NAME = as.character(NA), - ALT_FREQ = as.numeric(NA), - EFFECT = as.character(NA), - SYNONYMOUS = as.character(NA), - POS = as.numeric(NA), - ALT = as.character(NA), - NV_class = as.character(NA), - group = as.character(NA) -) - -# Create SNP variable and select useful variables -vcf <- vcf %>% - dplyr::select( - SAMPLE, - REGION, - VARIANT_NAME, - ALT_FREQ, - EFFECT, - SYNONYMOUS, - POS, - ALT - ) - -# Create dataframe with gene length for scheme -notation_empty <- data.frame( - gene = "", - len = 0 -) %>% - filter(len != 0) - -notation <- lapply( - names(coordinates), - function(x) { - add_row( - notation_empty, - gene = x, - len = length(coordinates[[x]]) - ) - } -) %>% - bind_rows() - -# Classifying variants -log_info("Classifying variants") -vcf <- vcf %>% - mutate( - NV_class = case_when( - str_detect(ALT, fixed("-")) | - str_detect(ALT, fixed("+")) ~ - "INDEL", - TRUE ~ "SNP" - ), - Class = case_when( - EFFECT == "intergenic_region" ~ "Intergenic", - TRUE ~ SYNONYMOUS - ), - POS = as.numeric(POS) - ) %>% - rowwise() %>% - mutate( - indel_len = case_when( - NV_class == "INDEL" ~ str_length(ALT) - 1 - ), - indel_class = case_when( - EFFECT == "intergenic_region" ~ "Intergenic", - NV_class == "INDEL" & - indel_len %% 3 == 0 ~ - "In frame", - NV_class == "INDEL" & - indel_len %% 3 > 0 ~ - "Frameshift" - ) - ) %>% - ungroup() %>% - mutate( - group = case_when( - EFFECT == "intergenic_region" ~ "Intergenic", - NV_class == "SNP" ~ Class, - NV_class == "INDEL" ~ indel_class - ) - ) - -# NSP data -npc <- read_csv(snakemake@input[["regions"]]) %>% - mutate( - summary_nsp = case_when( - region %in% paste("nsp", seq(4, 12, 1), sep = "") ~ "nsp4-12", - region %in% paste("nsp", seq(14, 16, 1), sep = "") ~ "nsp14-16", - TRUE ~ region - ), - summary_start = case_when( - region %in% paste("nsp", seq(4, 12, 1), sep = "") ~ 8555, - region %in% paste("nsp", seq(14, 16, 1), sep = "") ~ 18040, - TRUE ~ start - ), - summary_end = case_when( - region %in% paste("nsp", seq(4, 12, 1), sep = "") ~ 16236, - region %in% paste("nsp", seq(14, 16, 1), sep = "") ~ 21552, - TRUE ~ end - ) - ) %>% - filter(region != "nsp1") - - -# PLOTS -## SUMMARY FIGURE FOR WHOLE GENOME -log_info("Plotting summary figure for whole genome") - -if (nrow(vcf) == 0) { - log_warn("Whole-genome VCF has no rows") - vcf <- empty_vcf -} - -variants <- vcf %>% - filter(ALT_FREQ > 0) %>% - ggplot() + - aes( - x = POS, - y = factor(SAMPLE, date_order), - shape = factor(NV_class, c("SNP", "INDEL")), - color = group, - alpha = ALT_FREQ - ) + - geom_point(size = 3) + - geom_col( - data = notation, - aes( - x = len, - y = 0.3, - fill = factor(gene, rev(names(coordinates))) - ), - inherit.aes = FALSE, - width = 0.3 - ) + - scale_fill_manual(values = GENE_PALETTE) + - xlim(c(0, 29903)) + - scale_color_manual( - labels = NV_TYPE_NAMES, - values = NV_TYPE_PALETTE - ) + - scale_y_discrete(drop = FALSE) + - labs( - x = "SARS-CoV-2 genome position", - y = "Sample", - shape = "Variant class", - color = "Classification", - alpha = "Frequency", - fill = "Region" - ) + - guides( - fill = guide_legend(reverse = TRUE) - ) - -# Window plot -window_plot <- window %>% - ggplot() + - aes( - x = position, - y = fraction, - color = feature - ) + - geom_point() + - geom_line( - aes(group = 1), - colour = "black", - alpha = 0.3 - ) + - scale_y_continuous( - label = scales::percent, - limits = c(0, max(window$fraction) + 0.005) - ) + - xlim(c(0, 29903)) + - scale_color_manual(values = GENE_PALETTE) + - labs( - y = "Proportion of \n sites with NV", - x = "", - color = "Gen" - ) - -# Add nsp info -window_plot_nsp <- window_plot + - geom_vline( - data = npc, - aes(xintercept = summary_start), - color = "red" - ) + - geom_vline( - data = npc, - aes(xintercept = summary_end), - color = "red" - ) + - geom_text( - data = npc, - aes( - x = (summary_start + summary_end) / 2, - y = max(window$fraction) + 0.002, - label = summary_nsp - ), - inherit.aes = FALSE, - size = 5, - angle = 60 - ) - -figura <- ggarrange( - window_plot_nsp, - variants, - nrow = 2, - align = "v", - legend.grob = get_legend(variants), - heights = c(2, 6), - legend = "right", - labels = c("A", "B") -) - -ggsave( - filename = snakemake@output[["fig"]], - plot = figura, - width = 250, - height = 240, - units = "mm", - dpi = 250 -) - - -# Zoom in in spike -log_info("Plotting summary for variants in the spike") -spike_pos <- window %>% - filter(feature == "S") %>% - pull(position) - -vcf_spike <- vcf %>% - filter( - ALT_FREQ > 0, - POS %in% c(min(spike_pos):max(spike_pos)) - ) - -window_plot_spike <- window %>% - filter(feature == "S") %>% - ggplot() + - aes( - x = position, - y = fraction, - color = feature - ) + - geom_point() + - geom_line( - aes(group = 1), - colour = "black", - alpha = 0.3 - ) + - scale_y_continuous( - label = scales::percent, - limits = c(0, max(window$fraction) + 0.005) - ) + - xlim(c(min(spike_pos), max(spike_pos))) + - scale_color_manual( - values = GENE_PALETTE, - guide = "none" - ) + - labs( - y = "Proportion of \n sites with NV", - x = "" - ) - -if (nrow(vcf_spike) == 0) { - log_warn("Spike VCF has no rows") - vcf_spike <- empty_vcf -} - -variants_spike <- vcf_spike %>% - ggplot() + - aes( - x = POS, - y = factor(SAMPLE, date_order), - shape = factor(NV_class, c("SNP", "INDEL")), - color = group, - alpha = ALT_FREQ - ) + - geom_point(size = 3) + - xlim(c(min(spike_pos), max(spike_pos))) + - scale_color_manual( - labels = NV_TYPE_NAMES, - values = NV_TYPE_PALETTE - ) + - scale_y_discrete(drop = FALSE) + - labs( - x = "SARS-CoV-2 genome position", - y = "Sample", - shape = "Variant class", - color = "Classification", - alpha = "Frequency", - fill = "Region" - ) + - guides( - fill = guide_legend(reverse = TRUE) - ) - -figura_spike <- ggarrange( - window_plot_spike, - variants_spike, - nrow = 2, - align = "v", - legend.grob = get_legend(variants), - heights = c(2, 6), - legend = "right", - labels = c("A", "B") -) - -ggsave( - filename = snakemake@output[["fig_s"]], - plot = figura_spike, - width = 250, - height = 240, - units = "mm", - dpi = 250 -) - - -# Figure for no of heterozygous sites for each sample -log_info("Plotting no. of heterozygous sites for each sample") -figur_SNP_table <- vcf_snp %>% - filter(ALT_FREQ <= snakemake@params$max_alt_freq) %>% - left_join( - metadata, - by = c("SAMPLE" = "ID") - ) %>% - group_by(SAMPLE) %>% - summarise( - CollectionDate = min(as.Date(CollectionDate)), - n = n_distinct(POS) - ) %>% - ungroup() - -if (nrow(figur_SNP_table) == 0) { - log_warn("Filtered SNP table has no rows") - figur_SNP_table <- empty_vcf -} - -figur_SNP_time <- figur_SNP_table %>% - ggplot() + - aes( - x = CollectionDate, - y = n - ) + - geom_smooth( - method = "lm", - fill = "gray95", - alpha = 0.6 - ) + - geom_point() + - labs( - x = "Collection date", - y = "No. of polimorphic sites" - ) - -ggsave( - filename = snakemake@output[["fig_cor"]], - plot = figur_SNP_time, - width = 250, - height = 119.4, - units = "mm", - dpi = 250 -) - - -# PLOT TABLES -log_info("Saving plot tables") - -# Variants summary table -vcf %>% - select( - SAMPLE, - POS, - VARIANT_NAME, - ALT_FREQ, - NV_class, - group - ) %>% - rename( - sample = SAMPLE, - Variant = VARIANT_NAME, - Class = group - ) %>% - filter(ALT_FREQ > 0) %>% - mutate( - Class = case_when( - Class == "Yes" ~ "synonymous", - Class == "No" ~ "non_synonymous", - TRUE ~ Class - ) - ) %>% - write.csv(snakemake@output[["table_2"]], row.names = FALSE) - -# Window plot table -window %>% - transmute( - POS = position, - feature = feature, - prop_PolymorphicSites = fraction - ) %>% - write.csv(snakemake@output[["table_1"]], row.names = FALSE) - -# Heterozygous sites per sample table -vcf_snp %>% - filter(ALT_FREQ <= snakemake@params$max_alt_freq) %>% - left_join( - metadata, - by = c("SAMPLE" = "ID") - ) %>% - group_by(SAMPLE) %>% - summarise( - CollectionDate = min(as.Date(CollectionDate)), - n_PolymorphicSites = n_distinct(POS) - ) %>% - ungroup() %>% - rename(sample = SAMPLE) %>% - unique() %>% - write.csv(snakemake@output[["table_3"]], row.names = FALSE) - -# Stats for reporting -n_indels <- vcf %>% - filter(NV_class == "INDEL") %>% - pull(VARIANT_NAME) %>% - unique() %>% - length() - -n_snv <- length(unique(vcf$VARIANT_NAME)) - n_indels -model <- lm(n ~ CollectionDate, data = figur_SNP_table) - -# Calculate correlation, if possible -if (nrow(figur_SNP_table) > 2) { - p.value <- summary(model)$coefficients[2, 4] -} else { - p.value <- NA -} - -list( - "INDELS" = n_indels, - "SNV" = n_snv, - "window" = snakemake@params[["window"]], - "step" = snakemake@params[["step"]], - "r2" = summary(model)$r.squared[[1]], - "value" = ifelse(p.value < 0.001, "< 0.001", p.value) -) %>% - toJSON() %>% - write(snakemake@output[["json"]]) diff --git a/workflow/scripts/report/nv_panel_data.R b/workflow/scripts/report/nv_panel_data.R new file mode 100644 index 0000000..f49260f --- /dev/null +++ b/workflow/scripts/report/nv_panel_data.R @@ -0,0 +1,104 @@ +#!/usr/bin/env Rscript + +# Write stdout and stderr to log file +log <- file(snakemake@log[[1]], open = "wt") +sink(log, type = "message") +sink(log, type = "output") + +library(tidyverse) +library(jsonlite) +library(logger) +log_threshold(INFO) + +log_info("Reading variants") +variants <- read_delim( + snakemake@input$variants, + col_select = c( + "SAMPLE", + "REGION", + "POS", + "ALT", + "VARIANT_NAME", + "ALT_FREQ", + "EFFECT", + "SYNONYMOUS" + ) +) +log_debug("Read {nrow(variants)} rows") + +log_info("Reading dates from metadata") +dates <- read_delim( + snakemake@input$metadata, + col_select = c("ID", "CollectionDate") +) + +log_info("Dating variants") +variants <- left_join( + variants, dates, + by = c("SAMPLE" = "ID") +) + +log_info("Classifying variants") +variants <- variants %>% + mutate( + NV_class = ifelse( + str_detect(ALT, fixed("-")) | str_detect(ALT, fixed("+")), + "INDEL", + "SNP" + ), + Class = ifelse(EFFECT == "intergenic_region", "Intergenic", SYNONYMOUS), + POS = as.numeric(POS) + ) %>% + # rowwise() %>% + mutate( + indel_len = ifelse(NV_class == "INDEL", str_length(ALT) - 1, NA), + indel_class = case_when( + EFFECT == "intergenic_region" ~ "Intergenic", + (NV_class == "INDEL") & (indel_len %% 3 == 0) ~ "In frame", + (NV_class == "INDEL") & (indel_len %% 3 != 0) ~ "Frameshift" + ) + ) %>% + ungroup() %>% + mutate( + group = case_when( + EFFECT == "intergenic_region" ~ "Intergenic", + NV_class == "SNP" ~ Class, + NV_class == "INDEL" ~ indel_class + ) + ) %>% + filter(ALT_FREQ > 0) +log_debug("Processed {nrow(variants)} rows") + +if (nrow(variants) == 0) { + log_warning("No variants found, using an empty table") + variants <- tibble( + SAMPLE = date_order, + REGION = as.character(NA), + VARIANT_NAME = as.character(NA), + ALT_FREQ = as.numeric(NA), + EFFECT = as.character(NA), + SYNONYMOUS = as.character(NA), + POS = as.numeric(NA), + ALT = as.character(NA), + NV_class = as.character(NA), + group = as.character(NA) + ) +} + +log_info("Writing processed table") +write_csv(variants, snakemake@output$table) + +log_info("Writing NV summary") +nv_counts <- variants %>% + distinct(VARIANT_NAME, NV_class) %>% + count(NV_class) %>% + deframe() +list( + "INDELS" = nv_counts["INDEL"], + "SNV" = nv_counts["SNP"] +) %>% + write_json( + snakemake@output$json, + auto_unbox = TRUE, + digits = NA + ) diff --git a/workflow/scripts/report/nv_panel_plot.R b/workflow/scripts/report/nv_panel_plot.R new file mode 100644 index 0000000..07cc1b0 --- /dev/null +++ b/workflow/scripts/report/nv_panel_plot.R @@ -0,0 +1,160 @@ +#!/usr/bin/env Rscript + +# Write stdout and stderr to log file +log <- file(snakemake@log[[1]], open = "wt") +sink(log, type = "message") +sink(log, type = "output") + +library(tidyverse) +library(ggpubr) +library(jsonlite) +library(logger) +log_threshold(INFO) + +# Import file with plots style +source(snakemake@params[["design"]]) + +# Subplot A: windows ============================ +log_info("Reading variant windows") +window <- read_csv(snakemake@input$window) + +log_debug("Calculating X axis limits") +xlim_values <- c( + max(0, min(window$position) - snakemake@params$window_step), + max(window$position) + snakemake@params$window_step +) + +log_info("Plotting variant windows") +p1 <- window %>% + ggplot() + + aes( + x = position, + y = fraction, + color = feature + ) + + geom_point() + + geom_line( + aes(group = 1), + colour = "black", + alpha = 0.3 + ) + + scale_y_continuous( + label = scales::percent, + limits = c(0, max(window$fraction) + mean(window$fraction)) + ) + + xlim(xlim_values) + + # TODO: change GENE_PALETTE to selection of TRAJECTORY.PANEL.COLORS ? + scale_color_manual(values = GENE_PALETTE) + + labs( + y = "Proportion of\nsites with NV", + x = "", + color = "Gen" + ) + +log_info("Reading regions to highlight") +highlight <- read_csv(snakemake@input$highlight_window_regions) + +if (nrow(highlight) != 0) { + log_info("Highlighting {nrow(highlight)} regions") + highlighted_sites <- unique(c(highlight$start, highlight$end)) + p1 <- p1 + + geom_vline(xintercept = highlighted_sites, color = "orange") + + geom_text( + data = highlight, + aes( + x = (start + end) / 2, + y = max(window$fraction + mean(window$fraction) / 2), + label = region + ), + inherit.aes = FALSE, + size = 3, + angle = 45 + ) +} else { + log_info("No highlighted regions") +} + +# Subplot B: windows ============================ +log_info("Reading panel data") +panel <- read_csv(snakemake@input$panel) + +log_info("Calculating sample chronological order") +date_order <- panel %>% + arrange(CollectionDate) %>% + pull(SAMPLE) %>% + unique() + +log_info("Reading regions") +regions <- read_json(snakemake@input$regions, simplifyVector = TRUE) %>% + enframe(name = "region", value = "coords") %>% + mutate( + start = map_dbl(coords, 1), + end = map_dbl(coords, 2), + length = end - start + 1 + ) %>% + select(-coords) + +log_info("Plotting variants") +p2 <- panel %>% + ggplot() + + aes( + x = POS, + y = factor(SAMPLE, date_order), + shape = factor(NV_class, c("SNP", "INDEL")), + color = group, + alpha = ALT_FREQ + ) + + geom_point(size = 3) + + geom_col( + data = regions, + aes( + x = length, + y = 0.3, + # TODO: legend shows as NA those without match in `regions` + fill = factor(region, rev(regions$region)) + ), + inherit.aes = FALSE, + width = 0.3 + ) + + # TODO: change GENE_PALETTE to selection of TRAJECTORY.PANEL.COLORS ? + scale_fill_manual(values = GENE_PALETTE) + + xlim(xlim_values) + + scale_color_manual( + labels = NV_TYPE_NAMES, + values = NV_TYPE_PALETTE + ) + + scale_y_discrete(drop = FALSE) + + labs( + x = "Genome position", + y = "Sample", + shape = "Variant class", + color = "Classification", + alpha = "Frequency", + fill = "Region" + ) + + guides( + fill = guide_legend(reverse = TRUE) + ) + +# Plot arrangement ============================ +log_info("Arranging plots") +p <- ggarrange( + p1, + p2, + nrow = 2, + align = "v", + legend.grob = get_legend(p2), + heights = c(2, 6), + legend = "right", + labels = c("A", "B") +) + +log_info("Saving plot") +ggsave( + filename = snakemake@output$plot, + plot = p, + width = snakemake@params$plot_width_mm, + height = snakemake@params$plot_height_mm, + units = "mm", + dpi = 250 +) diff --git a/workflow/scripts/report/nv_panel_zoom_on_feature_data.py b/workflow/scripts/report/nv_panel_zoom_on_feature_data.py new file mode 100644 index 0000000..4989e68 --- /dev/null +++ b/workflow/scripts/report/nv_panel_zoom_on_feature_data.py @@ -0,0 +1,28 @@ +#!/usr/bin/env python3 + +import sys +import logging +import json + +import pandas as pd + + +if __name__ == "__main__": + + logging.basicConfig(filename=snakemake.log[0], format=snakemake.config["LOG_PY_FMT"], level=logging.INFO) + + logging.info("Reading regions") + with open(snakemake.input.regions) as f: + regions = json.load(f) + + if snakemake.wildcards.region_name not in regions: + logging.error(f"Region {snakemake.wildcards.region_name} is absent in {snakemake.input.regions}") + sys.exit(1) + + start, end = regions[snakemake.wildcards.region_name] + + logging.info("Reading input table") + df = pd.read_csv(snakemake.input.table) + + logging.info("Filtering sites and writing results") + df[(df.POS >= start) & (df.POS <= end)].to_csv(snakemake.output.table, index=False) diff --git a/workflow/scripts/report/window_zoom_on_feature_data.py b/workflow/scripts/report/window_zoom_on_feature_data.py new file mode 100644 index 0000000..36f1e9f --- /dev/null +++ b/workflow/scripts/report/window_zoom_on_feature_data.py @@ -0,0 +1,28 @@ +#!/usr/bin/env python3 + +import sys +import logging +import json + +import pandas as pd + + +if __name__ == "__main__": + + logging.basicConfig(filename=snakemake.log[0], format=snakemake.config["LOG_PY_FMT"], level=logging.INFO) + + logging.info("Reading regions") + with open(snakemake.input.regions) as f: + regions = json.load(f) + + if snakemake.wildcards.region_name not in regions: + logging.error(f"Region {snakemake.wildcards.region_name} is absent in {snakemake.input.regions}") + sys.exit(1) + + start, end = regions[snakemake.wildcards.region_name] + + logging.info("Reading input table") + df = pd.read_csv(snakemake.input.table) + + logging.info("Filtering sites and writing results") + df[(df.position >= start) & (df.position <= end)].to_csv(snakemake.output.table, index=False) diff --git a/workflow/scripts/window.py b/workflow/scripts/window.py index f5633ed..73233f8 100644 --- a/workflow/scripts/window.py +++ b/workflow/scripts/window.py @@ -1,6 +1,7 @@ #!/usr/bin/env python3 import logging +import json from typing import NewType, Dict, Iterable, List import pandas as pd @@ -135,6 +136,13 @@ def main(): logging.info("Saving results") windows.to_csv(snakemake.output.window_df, index=False) + logging.info("Saving window settings to JSON file") + with open(snakemake.output.json, "w") as fw: + json.dump( + {"window": snakemake.params.window, "step": snakemake.params.step}, + fw + ) + if __name__ == "__main__": main() From 16523fc4ff824672bb5b5025b86a418d9c9c8d1e Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Mon, 3 Nov 2025 12:40:19 +0100 Subject: [PATCH 098/138] Fix error for zero selected variants --- workflow/scripts/report/af_trajectory_panel_plot.R | 5 ++++- 1 file changed, 4 insertions(+), 1 deletion(-) diff --git a/workflow/scripts/report/af_trajectory_panel_plot.R b/workflow/scripts/report/af_trajectory_panel_plot.R index 0094068..d8f1d62 100644 --- a/workflow/scripts/report/af_trajectory_panel_plot.R +++ b/workflow/scripts/report/af_trajectory_panel_plot.R @@ -32,7 +32,10 @@ selected.colors <- sample(TRAJECTORY.PANEL.COLORS, length(selected.variants)) log_debug("Selected color: {selected.colors}") p <- variants %>% filter(VARIANT_NAME %in% selected.variants) %>% - mutate(gPOS = reorder(paste0("g.", POS), POS)) %>% + mutate( + gPOS = paste0("g.", POS), + gPOS = reorder(gPOS, POS) + ) %>% ggplot() + aes( x = interval, From 4d917426cfa69ae01010150e84674182da12f830 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Mon, 3 Nov 2025 13:19:16 +0100 Subject: [PATCH 099/138] Parametrize remaining plot size --- workflow/rules/report.smk | 12 +++++++++--- workflow/scripts/report/af_time_correlation_plot.R | 4 ++-- workflow/scripts/report/dnds_plots.R | 8 ++++---- .../report/polymorphic_sites_over_time_plot.R | 4 ++-- 4 files changed, 17 insertions(+), 11 deletions(-) diff --git a/workflow/rules/report.smk b/workflow/rules/report.smk index 29dfa4b..241711a 100644 --- a/workflow/rules/report.smk +++ b/workflow/rules/report.smk @@ -130,6 +130,8 @@ rule polymorphic_sites_over_time_plot: conda: "../envs/renv.yaml" params: design = config["PLOTS"], + plot_width_mm = 159.2, + plot_height_mm = 119.4, input: table = REPORT_DIR_PLOTS/"polymorphic_sites_over_time.csv", output: @@ -183,8 +185,8 @@ rule nv_panel_plot: params: design = config["PLOTS"], window_step = config["WINDOW"]["STEP"], - plot_height_mm = 250, - plot_width_mm = 240, + plot_height_mm = 250.0, + plot_width_mm = 240.0, input: panel = REPORT_DIR_TABLES/"nv_panel.csv", window = REPORT_DIR_TABLES/"window.csv", @@ -336,7 +338,9 @@ rule time_signal_plot: rule dnds_plots: conda: "../envs/renv.yaml" params: - design = config["PLOTS"] + design = config["PLOTS"], + plot_height_mm = 119.4, + plot_width_mm = 159.2, input: table = REPORT_DIR_TABLES/"dnds.csv", output: @@ -370,6 +374,8 @@ rule af_time_correlation_plot: conda: "../envs/renv.yaml" params: design = config["PLOTS"], + plot_height_mm = 119.4, + plot_width_mm = 159.2, input: correlations = REPORT_DIR_TABLES/"af_time_correlation.csv", output: diff --git a/workflow/scripts/report/af_time_correlation_plot.R b/workflow/scripts/report/af_time_correlation_plot.R index 87321cd..0d7bef4 100644 --- a/workflow/scripts/report/af_time_correlation_plot.R +++ b/workflow/scripts/report/af_time_correlation_plot.R @@ -43,8 +43,8 @@ p <- correlations %>% ggsave( filename = snakemake@output$plot, plot = p, - width = 159.2, - height = 119.4, + width = snakemake@params$plot_width_mm, + height = snakemake@params$plot_height_mm, units = "mm", dpi = 250 ) diff --git a/workflow/scripts/report/dnds_plots.R b/workflow/scripts/report/dnds_plots.R index f3aa651..ae44d98 100644 --- a/workflow/scripts/report/dnds_plots.R +++ b/workflow/scripts/report/dnds_plots.R @@ -53,8 +53,8 @@ p.dn.ds <- plot.df %>% ggsave( filename = snakemake@output$plot_dn_ds, plot = p.dn.ds, - width = 159.2, - height = 119.4, + width = snakemake@params$plot_width_mm, + height = snakemake@params$plot_height_mm, units = "mm", dpi = 250 ) @@ -78,8 +78,8 @@ p.omega <- plot.df %>% ggsave( filename = snakemake@output$plot_omega, plot = p.omega, - width = 159.2, - height = 119.4, + width = snakemake@params$plot_width_mm, + height = snakemake@params$plot_height_mm, units = "mm", dpi = 250 ) diff --git a/workflow/scripts/report/polymorphic_sites_over_time_plot.R b/workflow/scripts/report/polymorphic_sites_over_time_plot.R index 40564a3..91ba820 100644 --- a/workflow/scripts/report/polymorphic_sites_over_time_plot.R +++ b/workflow/scripts/report/polymorphic_sites_over_time_plot.R @@ -35,8 +35,8 @@ log_info("Saving plot") ggsave( filename = snakemake@output$plot, plot = p, - width = 250, - height = 119.4, + width = snakemake@params$plot_width_mm, + height = snakemake@params$plot_height_mm, units = "mm", dpi = 250 ) From fccdae87199349ec21b1305a93823d5ed42c6c22 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Mon, 3 Nov 2025 15:48:00 +0100 Subject: [PATCH 100/138] Update report font Change font from Montserrat to Noto Sans --- config/design_plots.R | 4 ++-- config/report.styles.css | 6 +++--- template.qmd | 4 ++-- 3 files changed, 7 insertions(+), 7 deletions(-) diff --git a/config/design_plots.R b/config/design_plots.R index 2eb17bd..b2055d4 100644 --- a/config/design_plots.R +++ b/config/design_plots.R @@ -8,13 +8,13 @@ showtext_auto(enable = FALSE) showtext_opts(dpi = 200) # Tema -font_add_google("Montserrat", "Montserrat") +font_add_google("Noto Sans", "Noto Sans") showtext_auto() theme_set(theme_minimal()) theme_update( - text = element_text(size = 16, family = "Montserrat"), + text = element_text(size = 16, family = "Noto Sans"), axis.title = element_text(size = 16), axis.line = element_line( linewidth = 0.5, diff --git a/config/report.styles.css b/config/report.styles.css index f81e0a9..6282f9a 100644 --- a/config/report.styles.css +++ b/config/report.styles.css @@ -9,15 +9,15 @@ } .title { - font-family: Montserrat; + font-family: "Noto Sans"; color: #FFFFFF } .quarto-title p { - font-family: Montserrat; + font-family: "Noto Sans"; color: #FFFFFF } html { - font-family: Montserrat; + font-family: "Noto Sans"; color: #000000; text-align: justify; } diff --git a/template.qmd b/template.qmd index 84fed59..d1b1dec 100644 --- a/template.qmd +++ b/template.qmd @@ -49,7 +49,7 @@ output-file: report.html - + @@ -143,7 +143,7 @@ table %>% columns = everything() ) %>% opt_table_font( - font = google_font("Montserrat") + font = google_font("Noto Sans") ) ``` From 9b2a2625f5e2112d5c7c45213929d20bcaedd1f6 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Mon, 3 Nov 2025 16:48:57 +0100 Subject: [PATCH 101/138] Update calculation of allele frequency-weighted distances Replace Python script with PathoGenOmics/afwdist, a faster, lightweight tool --- workflow/envs/afwdist.yaml | 5 + workflow/rules/distances.smk | 55 +++++-- workflow/scripts/extract_afwdist_variants.py | 90 ++++++++++ workflow/scripts/format_afwdist_results.py | 27 +++ workflow/scripts/weighted_distances.py | 163 ------------------- 5 files changed, 167 insertions(+), 173 deletions(-) create mode 100644 workflow/envs/afwdist.yaml create mode 100644 workflow/scripts/extract_afwdist_variants.py create mode 100644 workflow/scripts/format_afwdist_results.py delete mode 100644 workflow/scripts/weighted_distances.py diff --git a/workflow/envs/afwdist.yaml b/workflow/envs/afwdist.yaml new file mode 100644 index 0000000..8e29d0d --- /dev/null +++ b/workflow/envs/afwdist.yaml @@ -0,0 +1,5 @@ +channels: + - bioconda + - conda-forge +dependencies: + - afwdist==1.0.0 diff --git a/workflow/rules/distances.smk b/workflow/rules/distances.smk index d569bd2..833589a 100644 --- a/workflow/rules/distances.smk +++ b/workflow/rules/distances.smk @@ -1,17 +1,52 @@ -rule weighted_distances: - threads: 1 +rule extract_afwdist_variants: conda: "../envs/biopython.yaml" params: - samples = expand("{sample}", sample = iter_samples()), - mask_class = ["mask"] + sample_col = "SAMPLE", + position_col = "POS", + sequence_col = "ALT", + frequency_col = "ALT_FREQ", + mask_class = ["mask"], input: - tsv = OUTDIR/f"{OUTPUT_NAME}.variants.tsv", - vcf = lambda wildcards: select_problematic_vcf(), + variants = OUTDIR/f"{OUTPUT_NAME}.variants.tsv", + mask_vcf = lambda wildcards: select_problematic_vcf(), ancestor = OUTDIR/f"{OUTPUT_NAME}.ancestor.fasta", - reference = OUTDIR/"reference.fasta" + reference = OUTDIR/"reference.fasta", output: - distances = REPORT_DIR_TABLES/f"distances.csv" + variants = temp(OUTDIR/f"{OUTPUT_NAME}.variants.afwdist.csv"), log: - LOGDIR / "weighted_distances" / "log.txt" + LOGDIR/"extract_afwdist_variants"/"log.txt" script: - "../scripts/weighted_distances.py" + "../scripts/extract_afwdist_variants.py" + + +rule afwdist_weighted_distances: + conda: "../envs/afwdist.yaml" + params: + extra_args = "" + input: + variants = OUTDIR/f"{OUTPUT_NAME}.variants.afwdist.csv", + reference = OUTDIR/f"{OUTPUT_NAME}.ancestor.fasta", + output: + distances = temp(REPORT_DIR_TABLES/"distances.raw.csv"), + log: + LOGDIR/"afwdist_weighted_distances"/"log.txt" + shell: + "afwdist " + "-i {input.variants:q} " + "-r {input.reference:q} " + "-o {output.distances:q} " + "{params.extra_args} >{log:q} 2>&1" + + +rule format_afwdist_results: + conda: "../envs/biopython.yaml" + params: + samples = sorted(iter_samples()), + input: + distances = REPORT_DIR_TABLES/"distances.raw.csv", + output: + distances = REPORT_DIR_TABLES/"distances.csv", + log: + LOGDIR/"format_afwdist_results"/"log.txt" + script: + "../scripts/format_afwdist_results.py" diff --git a/workflow/scripts/extract_afwdist_variants.py b/workflow/scripts/extract_afwdist_variants.py new file mode 100644 index 0000000..7155a1b --- /dev/null +++ b/workflow/scripts/extract_afwdist_variants.py @@ -0,0 +1,90 @@ +#!/usr/bin/env python3 + +import logging +from typing import List + +import pandas as pd +from Bio import SeqIO +from Bio.SeqRecord import SeqRecord +from Bio.Seq import Seq + + +def read_monofasta(path: str) -> SeqRecord: + fasta = SeqIO.parse(path, "fasta") + record = next(fasta) + if next(fasta, None) is not None: + logging.warning(f"There are unread records left in '{path}'") + return record + + +def read_masked_sites(vcf_path: str, mask_classes: List[str]) -> List[int]: + """ + Parse a VCF containing positions for masking. Assumes the VCF file is + formatted as in: + github.com/W-L/ProblematicSites_SARS-CoV2/blob/master/problematic_sites_sarsCov2.vcf + with a "mask" or "caution" recommendation in column 7. + Masked sites are specified with params. + """ + vcf = pd.read_csv( + vcf_path, + sep="\s+", + comment="#", + names=("CHROM", "POS", "ID", "REF", "ALT", "QUAL", "FILTER", "INFO") + ) + return vcf.loc[vcf.FILTER.isin(mask_classes), "POS"].tolist() + + +def build_ancestor_variant_table(ancestor: Seq, reference: Seq, reference_name: str, masked_positions: List[int]) -> pd.DataFrame: + pos = [] + alt = [] + for i in range(1, len(ancestor) + 1): + if i not in masked_positions and ancestor[i-1] != reference[i-1]: + pos.append(i) + alt.append(reference[i-1]) + df = pd.DataFrame({snakemake.params.position_col: pos, snakemake.params.sequence_col: alt}) + df[snakemake.params.frequency_col] = 1 # As a reference genome, we assume all positions have fixed alleles + df[snakemake.params.sample_col] = reference_name + return df + + +if __name__ == "__main__": + + logging.basicConfig(filename=snakemake.log[0], format=snakemake.config["LOG_PY_FMT"], level=logging.INFO) + + colnames = { + snakemake.params.sample_col: "sample", + snakemake.params.position_col: "position", + snakemake.params.sequence_col: "sequence", + snakemake.params.frequency_col: "frequency" + } + + logging.info("Reading input tables") + # Variants + variants = pd.read_table(snakemake.input.variants, sep="\t") + logging.info(f"Read {len(variants)} variant records") + # VCF with sites to mask + masked_sites = read_masked_sites(snakemake.input.mask_vcf, snakemake.params.mask_class) + logging.info(f"Read {len(masked_sites)} masked positions") + + logging.info("Reading input FASTA files") + # Case ancestor + ancestor = read_monofasta(snakemake.input.ancestor) + logging.info(f"Ancestor: '{ancestor.description}', length={len(ancestor.seq)}") + # Alignment reference + reference = read_monofasta(snakemake.input.reference) + logging.info(f"Reference: '{reference.description}', length={len(reference.seq)}") + + logging.info("Processing ancestor variants") + ancestor_table = build_ancestor_variant_table(ancestor.seq, reference.seq, reference.id, masked_sites) + logging.info(f"Ancestor has {len(ancestor_table)} variants") + all_variants = pd.concat([variants, ancestor_table], ignore_index=True) + logging.info(f"Combined table has {len(all_variants)} variants") + + logging.info("Renaming and selecting columns") + output = all_variants.rename(columns=colnames)[list(colnames.values())] + logging.info("Filtering sites") + output = output[~output.position.isin(masked_sites)] + logging.info(f"There are {len(output)} rows left") + + logging.info("Writing results") + output.to_csv(snakemake.output.variants, index=False) diff --git a/workflow/scripts/format_afwdist_results.py b/workflow/scripts/format_afwdist_results.py new file mode 100644 index 0000000..ca9b24f --- /dev/null +++ b/workflow/scripts/format_afwdist_results.py @@ -0,0 +1,27 @@ +#!/usr/bin/env python3 + +import logging +import pandas as pd + + +if __name__ == "__main__": + + logging.basicConfig(filename=snakemake.log[0], format=snakemake.config["LOG_PY_FMT"], level=logging.INFO) + + logging.info("Read pairwise distances") + df = pd.read_csv(snakemake.input.distances) + + logging.info("Initializing formatted output") + output = pd.DataFrame( + columns=snakemake.params.samples, + index=snakemake.params.samples, + dtype="float64" + ) + + logging.info("Filling table") + for i, row in df.iterrows(): + output.loc[row.sample_m, row.sample_n] = row.distance + output.loc[row.sample_n, row.sample_m] = row.distance + + logging.info("Writing formatted results") + output.to_csv(snakemake.output.distances) diff --git a/workflow/scripts/weighted_distances.py b/workflow/scripts/weighted_distances.py deleted file mode 100644 index dbd8598..0000000 --- a/workflow/scripts/weighted_distances.py +++ /dev/null @@ -1,163 +0,0 @@ -#!/usr/bin/env python3 -# -*- coding: utf-8 -*- - -# Adapted script from https://github.com/PathoGenOmics-Lab/genetic-distances - -import logging -from typing import List, Tuple - -import pandas as pd -from Bio import SeqIO -from Bio.SeqRecord import SeqRecord -from Bio.Seq import Seq - - -def read_monofasta(path: str) -> SeqRecord: - fasta = SeqIO.parse(path, "fasta") - record = next(fasta) - if next(fasta, None) is not None: - logging.warning(f"There are unread records left in '{path}'") - return record - - -def read_masked_sites(vcf_path: str, mask_classes: List[str]) -> List[int]: - """ - Parse a VCF containing positions for masking. Assumes the VCF file is - formatted as in: - github.com/W-L/ProblematicSites_SARS-CoV2/blob/master/problematic_sites_sarsCov2.vcf - with a "mask" or "caution" recommendation in column 7. - Masked sites are specified with params. - """ - vcf = pd.read_csv( - vcf_path, - sep="\s+", - comment="#", - names=("CHROM", "POS", "ID", "REF", "ALT", "QUAL", "FILTER", "INFO") - ) - return vcf.loc[vcf.FILTER.isin(mask_classes), "POS"].tolist() - - -def build_ancestor_variant_table(ancestor: Seq, reference: Seq, reference_name: str, masked_positions: List[int]) -> pd.DataFrame: - pos = [] - alt = [] - for i in range(1, len(ancestor) + 1): - if i not in masked_positions and ancestor[i-1] != reference[i-1]: - pos.append(i) - alt.append(reference[i-1]) - df = pd.DataFrame({"POS": pos, "ALT": alt}) - df["ALT_FREQ"] = 1 # As a reference genome, we assume all positions are monomorphic - df["SAMPLE"] = reference_name - return df - - -def get_frequencies_in_position(variant_table: pd.DataFrame, sample_name: str, position: int, reference: Seq) -> Tuple[float]: - frequencies = {alt: 0. for alt in variant_table["ALT"].unique()} - alt_keys = sorted(frequencies.keys()) - sample_variant_table = variant_table[variant_table["SAMPLE"] == sample_name] - # If the position has polimorphisims, allele frequencies are captured - if position in sample_variant_table["POS"].values: - alleles = sample_variant_table[sample_variant_table["POS"] == position] - for base in alt_keys: - if base in alleles["ALT"].values: - frequencies[base] = float(alleles["ALT_FREQ"][alleles["ALT"] == base].iloc[0]) - # Obtain frequency for reference allele - reference_allele = reference[position-1] - reference_freq = 1 - sum(frequencies.values()) - if reference_allele in frequencies: - frequencies[reference[position-1]] += reference_freq - else: - frequencies[reference[position-1]] = reference_freq - return tuple(frequencies[base] for base in alt_keys) - - -def heterozygosity(freqs: Tuple[float]) -> float: - return 1 - sum([f ** 2 for f in freqs]) - - -def calc_fst_weir_cockerham(hs: float, ht: float) -> float: - return (ht - hs) / ht if ht != 0 else 0 - - -def build_cache(variant_table: pd.DataFrame, samples: List[str], reference: Seq): - cache = {"freq": {}, "hz": {}} - for sample_name in set(samples): - for position in variant_table["POS"].astype("Int64").unique(): - if sample_name not in cache["freq"]: - cache["freq"][sample_name] = {} - if sample_name not in cache["hz"]: - cache["hz"][sample_name] = {} - cache["freq"][sample_name][position] = get_frequencies_in_position(variant_table, sample_name, position, reference) - logging.debug(f"Frequencies for '{sample_name}':{position} = {cache['freq'][sample_name][position]}") - cache["hz"][sample_name][position] = heterozygosity(cache["freq"][sample_name][position]) - logging.debug(f"Heterozygosity for '{sample_name}':{position} = {cache['hz'][sample_name][position]}") - return cache - - -def calc_heterozygosities(sample1_name: str, sample2_name: str, pos: int, cache: dict): - logging.debug(f"Calculating heterozygosities at position {pos} for '{sample1_name}' and '{sample2_name}'") - # Retrieve pre-computed values - freqs1 = cache["freq"][sample1_name][pos] - freqs2 = cache["freq"][sample2_name][pos] - hs1 = cache["hz"][sample1_name][pos] - hs2 = cache["hz"][sample2_name][pos] - # Calculate pairwise values - total_freqs = [(f1 + f2) / 2 for f1, f2 in zip(freqs1, freqs2)] - ht = heterozygosity(total_freqs) - hs = (hs1 + hs2) / 2 - return hs, ht - - -def calculate_pairwise_distance(positions: List[int], sample1_name: str, sample2_name: str, cache: dict) -> float: - if len(positions) == 0: - return 0 - else: - return sum([calc_fst_weir_cockerham(*calc_heterozygosities(sample1_name, sample2_name, pos, cache)) for pos in positions]) - - -def calculate_sample_distances(positions: List[int], sample_name: str, samples: List[str], cache: dict) -> List[float]: - return [calculate_pairwise_distance(positions, sample_name, other_sample_name, cache) for other_sample_name in samples] - - -def calculate_distance_matrix(variant_table: pd.DataFrame, samples: List[str], reference: Seq) -> pd.DataFrame: - positions = variant_table["POS"].astype("Int64").unique().tolist() - cache = build_cache(variant_table, samples, reference) - distance_matrix = {} - for sample_name in samples: - distance_matrix[sample_name] = calculate_sample_distances(positions, sample_name, samples, cache) - for i in range(len(samples)): - for j in range(i+1, len(samples)): - distance_matrix[samples[j]][i] = distance_matrix[samples[i]][j] - return pd.DataFrame(distance_matrix, index=samples) - - -def main(): - - logging.basicConfig(filename=snakemake.log[0], format=snakemake.config["LOG_PY_FMT"], level=logging.INFO) - - logging.info("Reading input FASTA files") - ancestor = read_monofasta(snakemake.input.ancestor) - logging.debug(f"Ancestor: '{ancestor.description}', length={len(ancestor.seq)}") - reference = read_monofasta(snakemake.input.reference) - logging.debug(f"Reference: '{reference.description}', length={len(reference.seq)}") - - logging.info("Reading input tables") - masked_positions = read_masked_sites(snakemake.input.vcf, snakemake.params.mask_class) - logging.debug(f"Read {len(masked_positions)} masked positions") - input_table = pd.read_table(snakemake.input.tsv, sep="\t") - logging.debug(f"Read {len(input_table)} rows in input TSV") - ancestor_table = build_ancestor_variant_table(ancestor.seq, reference.seq, reference.id, masked_positions) - logging.debug(f"Ancestor has {len(ancestor_table)} variants") - variant_table = pd.concat([input_table, ancestor_table], ignore_index=True) - logging.debug(f"Combined table has {len(variant_table)} variants") - - logging.info(f"Calculating distance matrix") - sample_names = snakemake.params.samples + [reference.id] - distances = calculate_distance_matrix(variant_table, sample_names, ancestor.seq) - logging.debug(f"Distance matrix has shape: {distances.shape}") - - logging.info("Writing results") - distances.to_csv(snakemake.output.distances) - - -if __name__ == "__main__": - main() From 4186a4c301bc29fadf31cc6317430364c0b30fc9 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Mon, 3 Nov 2025 17:18:18 +0100 Subject: [PATCH 102/138] Remove unnecessary R dependencies --- workflow/envs/renv.yaml | 2 -- 1 file changed, 2 deletions(-) diff --git a/workflow/envs/renv.yaml b/workflow/envs/renv.yaml index 43568d8..ceb6a99 100644 --- a/workflow/envs/renv.yaml +++ b/workflow/envs/renv.yaml @@ -5,7 +5,6 @@ dependencies: - r-base=4.3.2 - r-tidyverse==2.0.0 - r-ggrepel==0.9.3 - - r-stringi==1.7.12 - r-ggpubr==0.6.0 - bioconductor-ggtree==3.10.0 - r-ape==5.8 @@ -15,5 +14,4 @@ dependencies: - r-future.apply==1.11.0 - r-scales==1.3.0 - r-showtext==0.9_6 - - r-jsonlite==1.8.5 - r-logger==0.2.2 From 8352ce31188984cb62003c41f80df6db15d77b8c Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Mon, 3 Nov 2025 17:59:51 +0100 Subject: [PATCH 103/138] Resize trajectory correlation hierarchical clustering heatmap Sets source editor as defaults well --- template.qmd | 20 ++++++++++++-------- 1 file changed, 12 insertions(+), 8 deletions(-) diff --git a/template.qmd b/template.qmd index d1b1dec..d0772bc 100644 --- a/template.qmd +++ b/template.qmd @@ -16,7 +16,7 @@ format: toc-title: Summary number-sections: true css: config/report.styles.css -editor: visual +editor: source params: ufboot_reps: "" shalrt_reps: "" @@ -169,7 +169,7 @@ $`r stats[["boot"]]`$% and a SH-aLRT score of $`r stats[["alrt"]]`$% (@fig-tree_ ![Maximum-likelihood phylogeny with $`r params$ufboot_reps`$ UFBoot and $`r params$shalrt_reps`$ SH-aLRT support replicates of the target dataset and its context samples. The clade that contains the target -samples is squared in red.](`r params$tree_ml`){#fig-tree_ml} +samples is squared.](`r params$tree_ml`){#fig-tree_ml} ### Nucleotide diversity comparison @@ -238,8 +238,8 @@ The correlation of the allele frequency of each NV with the time since the initial sampling has been calculated (@fig-volcano). ![`r cor.method.name` correlation coefficients and adjusted p-values of -allele frequencies with time. Red dashed line indicates adjusted $p = 0.05$. -Labeled dots represent nucleotide variants correlated with time +allele frequencies with time. The dashed line indicates adjusted (false discovery rate) $p = 0.05$. +Labeled dots represent nucleotide variants whose allele frequency is correlated with time (adjusted $p < 0.05$).](`r params$volcano`){#fig-volcano} Significantly correlated nucleotide variants are described in more detail in @fig-panel. @@ -262,8 +262,8 @@ $`r correlation`$ and a p-value of $`r p_value_lm`$. The estimated evolutionary rate is $`r sub_rate`$ number of changes per year (@fig-tempest). ![Scatterplot depicting the relationship between root-to-tip -distances and the number of days passed since the first sample. The red -line shows the linear model fit.](`r params$tempest`){#fig-tempest} +distances and the number of days passed since the first sample. The solid +line represents the linear model fit.](`r params$tempest`){#fig-tempest} ### Correlation between alternative alleles @@ -273,13 +273,17 @@ zooming in on specific regions. ```{r heatmap, echo = F, message = F, fig.align = 'center'} #| label: fig-heatmap -#| fig-cap: "Interactive hierarchically clustered heatmap of the pairwise correlation coefficients between the time series of allele frequencies in the case study." +#| fig-width: 6.27 +#| fig-height: 6.27 +#| fig-cap: "Interactive hierarchically clustered heatmap of the pairwisecorrelation coefficients between the time series of allele frequencies in the case study." heatmaply_cor( cor.mat, fontsize_row = 8, fontsize_col = 8, - column_text_angle = 45 + column_text_angle = 45, + width = 600, + height = 600 ) ``` From 245ba2328473e44abd18984fca6ede949dd4df16 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Mon, 3 Nov 2025 18:33:57 +0100 Subject: [PATCH 104/138] Harmonize plot colors and shapes --- .../scripts/report/af_time_correlation_plot.R | 11 ++- .../scripts/report/context_phylogeny_data.R | 89 ++++++++++--------- .../scripts/report/context_phylogeny_plot.R | 2 +- .../report/polymorphic_sites_over_time_plot.R | 2 +- workflow/scripts/report/time_signal_plot.R | 4 +- 5 files changed, 58 insertions(+), 50 deletions(-) diff --git a/workflow/scripts/report/af_time_correlation_plot.R b/workflow/scripts/report/af_time_correlation_plot.R index 0d7bef4..9ee9a19 100644 --- a/workflow/scripts/report/af_time_correlation_plot.R +++ b/workflow/scripts/report/af_time_correlation_plot.R @@ -27,13 +27,18 @@ p <- correlations %>% x = coefficient, y = trans.p ) + - geom_point() + - geom_text_repel(aes(label = label), max.overlaps = 1000, direction = "x") + + geom_text_repel(aes(label = label), max.overlaps = 10000, direction = "x") + + geom_point( + data = function(x) subset(x, !is.na(label)), + color = "orange", + size = 2 + ) + + geom_point(size = 2, shape = 1) + xlim(c(-1, 1)) + geom_hline( aes(yintercept = -log10(0.05)), linetype = 2, - color = "red" + color = "orange" ) + labs( x = "Correlation coefficient", diff --git a/workflow/scripts/report/context_phylogeny_data.R b/workflow/scripts/report/context_phylogeny_data.R index 5e5eb12..704df80 100644 --- a/workflow/scripts/report/context_phylogeny_data.R +++ b/workflow/scripts/report/context_phylogeny_data.R @@ -8,7 +8,6 @@ sink(log, type = "output") library(tidyverse) library(jsonlite) library(ape) -library(ggtree) library(logger) log_threshold(INFO) @@ -23,40 +22,44 @@ TREE_LEGEND_NAMES["boot_alrt_pass"] <- sprintf( snakemake@params[["alrt_th"]], "%" ) +log_info("Reading tree") tree_ml <- read.tree(snakemake@input[["tree"]]) %>% - root( - snakemake@params[["ref_name"]], - resolve.root = TRUE - ) + root( + snakemake@params[["ref_name"]], + resolve.root = TRUE + ) +log_debug("Read tree with {length(tree_ml$node.label)} labels") +log_info("Reading target names from FASTA") target_names <- read.dna( - snakemake@input[["target_fasta"]], - format = "fasta", - as.matrix = FALSE, + snakemake@input[["target_fasta"]], + format = "fasta", + as.matrix = FALSE, ) +log_debug("Read {length(target_names)} records") +log_info("Processing target names") target_names <- target_names[ - !startsWith(names(target_names), snakemake@config[["ALIGNMENT_REFERENCE"]]) + !startsWith(names(target_names), snakemake@config[["ALIGNMENT_REFERENCE"]]) ] target_names <- names(target_names) +log_debug("{length(target_names)} records remaining after processing") # ML tree with context data # Internal nodes color # Node labels contain SH-aLRT/UFboot values +log_info("Reading support values from labels") +labels <- strsplit(tree_ml$node.label, "/") aLRT.values <- sapply( - strsplit(tree_ml$node.label, "/"), - function(x) { - as.numeric(x[1]) - } + labels, + function(x) as.numeric(x[1]) ) - bootstrap.values <- sapply( - strsplit(tree_ml$node.label, "/"), - function(x) { - as.numeric(x[2]) - } + labels, + function(x) as.numeric(x[2]) ) +log_info("Calculating support mask for the given thresholds") aLRT.mask <- aLRT.values >= snakemake@params[["alrt_th"]] boot.mask <- bootstrap.values >= snakemake@params[["boot_th"]] @@ -73,15 +76,15 @@ tree_ml.labels <- tree_ml$tip.label[1:tree_ml.nodes] tree_ml.node.pass <- c(rep(FALSE, tree_ml.ntips), aLRT.mask & boot.mask) ml.tree.annot <- tibble( - node = 1:tree_ml.nodes, + node = 1:tree_ml.nodes, ) %>% - mutate( - Class = case_when( - tree_ml.labels %in% target_names ~ TREE_LEGEND_NAMES["tip_label"], - tree_ml.node.pass ~ TREE_LEGEND_NAMES["boot_alrt_pass"], - TRUE ~ NA - ) + mutate( + Class = case_when( + tree_ml.labels %in% target_names ~ TREE_LEGEND_NAMES["tip_label"], + tree_ml.node.pass ~ TREE_LEGEND_NAMES["boot_alrt_pass"], + TRUE ~ NA ) + ) # Write output files log_info("Writing tree annotation") @@ -90,22 +93,22 @@ write_csv(ml.tree.annot, snakemake@output$annotation) log_info("Writing JSON data") target.node <- tree_ml$node.label[target.mrca - length(tree_ml$tip.label)] list( - "boot" = strsplit(target.node, "/")[[1]][2] %>% as.numeric(), - "alrt" = strsplit(target.node, "/")[[1]][1] %>% as.numeric(), - "monophyly" = ifelse( - is.monophyletic(tree_ml, target_names), - "are", - "are not" - ), - "target_mrca" = target.mrca, - "clade_tips" = target.mrca.clade.ntips, - "max_tip_length" = max(node.depth.edgelength(tree_ml)[ - 1:length(tree_ml$tip.label) - ]), - "root" = snakemake@params[["ref_name"]] + "boot" = strsplit(target.node, "/")[[1]][2] %>% as.numeric(), + "alrt" = strsplit(target.node, "/")[[1]][1] %>% as.numeric(), + "monophyly" = ifelse( + is.monophyletic(tree_ml, target_names), + "are", + "are not" + ), + "target_mrca" = target.mrca, + "clade_tips" = target.mrca.clade.ntips, + "max_tip_length" = max(node.depth.edgelength(tree_ml)[ + 1:length(tree_ml$tip.label) + ]), + "root" = snakemake@params[["ref_name"]] ) %>% - write_json( - snakemake@output$json, - auto_unbox = TRUE, - digits = NA - ) + write_json( + snakemake@output$json, + auto_unbox = TRUE, + digits = NA + ) diff --git a/workflow/scripts/report/context_phylogeny_plot.R b/workflow/scripts/report/context_phylogeny_plot.R index dd6baf8..8b56164 100644 --- a/workflow/scripts/report/context_phylogeny_plot.R +++ b/workflow/scripts/report/context_phylogeny_plot.R @@ -39,7 +39,7 @@ annotation <- read_csv(snakemake@input$annotation) log_info("Plotting M-L tree with context samples") p <- ggtree(tree_ml, layout = "circular") %<+% annotation + - geom_highlight(node = json$target_mrca, colour = "red", alpha = 0) + + geom_highlight(node = json$target_mrca, colour = "orange", alpha = 0) + geom_point(aes(color = Class, size = Class)) + geom_treescale(1.05 * json$max_tip_length) + geom_rootedge(0.05 * json$max_tip_length) + diff --git a/workflow/scripts/report/polymorphic_sites_over_time_plot.R b/workflow/scripts/report/polymorphic_sites_over_time_plot.R index 91ba820..83c1425 100644 --- a/workflow/scripts/report/polymorphic_sites_over_time_plot.R +++ b/workflow/scripts/report/polymorphic_sites_over_time_plot.R @@ -25,7 +25,7 @@ p <- df %>% alpha = 0.6, colour = "orange" ) + - geom_point(size = 1, shape = 1) + + geom_point(size = 2, shape = 1) + labs( x = "Days since the initial sampling", y = "No. of polimorphic sites" diff --git a/workflow/scripts/report/time_signal_plot.R b/workflow/scripts/report/time_signal_plot.R index d13299a..27741f0 100644 --- a/workflow/scripts/report/time_signal_plot.R +++ b/workflow/scripts/report/time_signal_plot.R @@ -29,9 +29,9 @@ p <- time.signal %>% method = "lm", fill = "gray95", alpha = 0.6, - color = "red" + color = "orange" ) + - geom_point() + + geom_point(size = 2, shape = 1) + labs( y = "Root-to-tip distance", x = "Days since the initial sampling" From c30ba2c49ff22539a19fce5a321eb71a708ac5b9 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Mon, 3 Nov 2025 18:39:13 +0100 Subject: [PATCH 105/138] Add seed param to IQTREE rules Also updates the style of the shell directive and adds -nm where relevant --- workflow/rules/asr.smk | 13 ++++++------- workflow/rules/context.smk | 23 +++++++++++------------ 2 files changed, 17 insertions(+), 19 deletions(-) diff --git a/workflow/rules/asr.smk b/workflow/rules/asr.smk index 5acaded..7bc1c8d 100644 --- a/workflow/rules/asr.smk +++ b/workflow/rules/asr.smk @@ -2,6 +2,7 @@ rule reconstruct_ancestral_sequence: threads: 4 conda: "../envs/iqtree.yaml" params: + seed = 7291, seqtype = "DNA", name = OUTPUT_NAME, outgroup = config["ALIGNMENT_REFERENCE"], @@ -14,13 +15,11 @@ rule reconstruct_ancestral_sequence: log: LOGDIR / "reconstruct_ancestral_sequence" / "log.txt" shell: - """ - mkdir -p {output.folder} - iqtree2 \ - -asr \ - -o {params.outgroup} -T AUTO --threads-max {threads} -s {input.fasta} \ - --seqtype {params.seqtype} -m {params.model} --prefix {output.folder}/{params.name} >{log} 2>&1 - """ + "mkdir -p {output.folder} && " + "iqtree2 -seed {params.seed} " + "-asr " + "-o {params.outgroup} -T AUTO --threads-max {threads} -s {input.fasta} " + "--seqtype {params.seqtype} -m {params.model} --prefix {output.folder}/{params.name} >{log} 2>&1" rule ancestor_fasta: diff --git a/workflow/rules/context.smk b/workflow/rules/context.smk index 3bb2355..9f8fa05 100644 --- a/workflow/rules/context.smk +++ b/workflow/rules/context.smk @@ -74,12 +74,14 @@ rule ml_context_tree: conda: "../envs/iqtree.yaml" shadow: "shallow" params: + seed = 7291, seqtype = "DNA", name = OUTPUT_NAME, ufboot = config["UFBOOT"]["REPS"], alrt = config["SHALRT"]["REPS"], outgroup = config["ALIGNMENT_REFERENCE"], - model = config["TREE_MODEL"] + model = config["TREE_MODEL"], + max_iterations_convergence = 1000, input: fasta = OUTDIR/"nextalign"/f"{OUTPUT_NAME}.aligned.masked.fasta", outgroup_aln = OUTDIR/"context"/"nextalign"/"context_sequences.aligned.masked.fasta" @@ -89,14 +91,11 @@ rule ml_context_tree: log: LOGDIR / "ml_context_tree" / "log.txt" shell: - """ - exec >{log} - exec 2>&1 - - awk '/^>/{{p=seen[$0]++}}!p' {input.fasta} {input.outgroup_aln} > aln.fasta - mkdir -p {output.folder} - iqtree2 \ - -B {params.ufboot} -alrt {params.alrt} \ - -o {params.outgroup} -T AUTO --threads-max {threads} -s aln.fasta \ - --seqtype {params.seqtype} -m {params.model} --prefix {output.folder}/{params.name} - """ + "exec >{log} && exec 2>&1; " + "awk '/^>/{{p=seen[$0]++}}!p' {input.fasta} {input.outgroup_aln} >aln.fasta && " + "mkdir -p {output.folder} && " + "iqtree2 -seed {params.seed} " + "-bb {params.ufboot} -alrt {params.alrt} " + "-o {params.outgroup} -T AUTO --threads-max {threads} -s aln.fasta " + "-nm {params.max_iterations_convergence} " + "--seqtype {params.seqtype} -m {params.model} --prefix {output.folder}/{params.name}" From e2f260275b6baabf872bd470b68816c725f5b17b Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 4 Nov 2025 10:40:11 +0100 Subject: [PATCH 106/138] Set explicit afwdist input dtypes --- workflow/scripts/extract_afwdist_variants.py | 15 ++++++++++++++- 1 file changed, 14 insertions(+), 1 deletion(-) diff --git a/workflow/scripts/extract_afwdist_variants.py b/workflow/scripts/extract_afwdist_variants.py index 7155a1b..6a7bf3f 100644 --- a/workflow/scripts/extract_afwdist_variants.py +++ b/workflow/scripts/extract_afwdist_variants.py @@ -47,6 +47,14 @@ def build_ancestor_variant_table(ancestor: Seq, reference: Seq, reference_name: return df +DTYPES = { + "sample": "object", + "position": "int64", + "sequence": "object", + "frequency": "float64" +} + + if __name__ == "__main__": logging.basicConfig(filename=snakemake.log[0], format=snakemake.config["LOG_PY_FMT"], level=logging.INFO) @@ -81,7 +89,12 @@ def build_ancestor_variant_table(ancestor: Seq, reference: Seq, reference_name: logging.info(f"Combined table has {len(all_variants)} variants") logging.info("Renaming and selecting columns") - output = all_variants.rename(columns=colnames)[list(colnames.values())] + output = ( + all_variants + .rename(columns=colnames) + [list(colnames.values())] + .astype(DTYPES) + ) logging.info("Filtering sites") output = output[~output.position.isin(masked_sites)] logging.info(f"There are {len(output)} rows left") From 2dba19f71e9572152bcc211e1f4d15de37acef65 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 4 Nov 2025 10:55:17 +0100 Subject: [PATCH 107/138] Add --keep-going to tests --- .github/workflows/test_apptainer.yml | 2 +- .github/workflows/test_v7.yml | 2 +- .github/workflows/test_v8.yml | 2 +- .github/workflows/test_v9.yml | 2 +- 4 files changed, 4 insertions(+), 4 deletions(-) diff --git a/.github/workflows/test_apptainer.yml b/.github/workflows/test_apptainer.yml index d70f359..08b8f26 100644 --- a/.github/workflows/test_apptainer.yml +++ b/.github/workflows/test_apptainer.yml @@ -26,7 +26,7 @@ jobs: environment-file: .test/environment_apptainer.yaml - name: Run test pipeline shell: bash -el {0} - run: snakemake --snakefile .test/Snakefile --configfile config/config.yaml .test/targets.yaml -c1 --sdm apptainer conda + run: snakemake --snakefile .test/Snakefile --configfile config/config.yaml .test/targets.yaml -c1 --sdm apptainer conda --keep-going - name: Pack logs if: success() || failure() shell: bash -el {0} diff --git a/.github/workflows/test_v7.yml b/.github/workflows/test_v7.yml index 93c6a4e..fba8bca 100644 --- a/.github/workflows/test_v7.yml +++ b/.github/workflows/test_v7.yml @@ -26,7 +26,7 @@ jobs: environment-file: .test/environment_v7.yaml - name: Run test pipeline shell: bash -el {0} - run: snakemake --snakefile .test/Snakefile --configfile config/config.yaml .test/targets.yaml --use-conda -c1 --conda-frontend conda + run: snakemake --snakefile .test/Snakefile --configfile config/config.yaml .test/targets.yaml --use-conda -c1 --conda-frontend conda --keep-going - name: Pack logs if: success() || failure() shell: bash -el {0} diff --git a/.github/workflows/test_v8.yml b/.github/workflows/test_v8.yml index 0043c36..4f4af1d 100644 --- a/.github/workflows/test_v8.yml +++ b/.github/workflows/test_v8.yml @@ -26,7 +26,7 @@ jobs: environment-file: .test/environment_v8.yaml - name: Run test pipeline shell: bash -el {0} - run: snakemake --snakefile .test/Snakefile --configfile config/config.yaml .test/targets.yaml --use-conda -c1 --conda-frontend conda + run: snakemake --snakefile .test/Snakefile --configfile config/config.yaml .test/targets.yaml --use-conda -c1 --conda-frontend conda --keep-going - name: Pack logs if: success() || failure() shell: bash -el {0} diff --git a/.github/workflows/test_v9.yml b/.github/workflows/test_v9.yml index 1f5371a..1845334 100644 --- a/.github/workflows/test_v9.yml +++ b/.github/workflows/test_v9.yml @@ -26,7 +26,7 @@ jobs: environment-file: .test/environment_v9.yaml - name: Run test pipeline shell: bash -el {0} - run: snakemake --snakefile .test/Snakefile --configfile config/config.yaml .test/targets.yaml -c1 --sdm conda + run: snakemake --snakefile .test/Snakefile --configfile config/config.yaml .test/targets.yaml -c1 --sdm conda --keep-going - name: Pack logs if: success() || failure() shell: bash -el {0} From 69a4b52318e1eb6d60012599b493717b77bc430f Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 4 Nov 2025 15:43:45 +0100 Subject: [PATCH 108/138] Update R library imports Avoids loading the whole tidyverse when unnecessary --- config/design_plots.R | 2 +- workflow/scripts/compile_variants.R | 16 +-- workflow/scripts/compile_vcf_fields_longer.R | 18 ++-- workflow/scripts/dnds_data.R | 81 +++++++------- workflow/scripts/download_context.R | 23 ++-- workflow/scripts/filter_tsv.R | 5 +- workflow/scripts/format_vcf_fields_longer.R | 101 +++++++++--------- workflow/scripts/merge_annotation.R | 4 +- .../scripts/report/af_time_correlation_data.R | 5 +- .../scripts/report/af_time_correlation_plot.R | 4 +- .../scripts/report/af_trajectory_panel_plot.R | 8 +- .../scripts/report/allele_freq_tree_data.R | 4 +- .../scripts/report/allele_freq_tree_plot.R | 4 +- .../scripts/report/context_phylogeny_data.R | 4 +- .../scripts/report/context_phylogeny_plot.R | 4 +- workflow/scripts/report/demix_plot.R | 5 +- workflow/scripts/report/demix_plot_data.R | 4 +- workflow/scripts/report/diversity_data.R | 4 +- workflow/scripts/report/diversity_plot.R | 5 +- workflow/scripts/report/dnds_plots.R | 6 +- workflow/scripts/report/heatmap.R | 10 +- workflow/scripts/report/nv_panel_data.R | 6 +- workflow/scripts/report/nv_panel_plot.R | 7 +- .../report/polymorphic_sites_over_time_data.R | 4 +- .../report/polymorphic_sites_over_time_plot.R | 5 +- workflow/scripts/report/summary_table.R | 3 +- workflow/scripts/report/time_signal_data.R | 4 +- workflow/scripts/report/time_signal_plot.R | 4 +- workflow/scripts/summarise_demix.R | 6 +- 29 files changed, 214 insertions(+), 142 deletions(-) diff --git a/config/design_plots.R b/config/design_plots.R index b2055d4..fc63584 100644 --- a/config/design_plots.R +++ b/config/design_plots.R @@ -1,6 +1,6 @@ # Jordi Sevilla -library(tidyverse) +library(ggplot2) library(showtext) # Ajustes #### diff --git a/workflow/scripts/compile_variants.R b/workflow/scripts/compile_variants.R index 7e7baaa..a3f435a 100644 --- a/workflow/scripts/compile_variants.R +++ b/workflow/scripts/compile_variants.R @@ -5,19 +5,21 @@ log <- file(snakemake@log[[1]], open = "wt") sink(log, type = "message") sink(log, type = "output") -library(tidyverse) +library(readr) +library(dplyr) library(logger) + log_threshold(INFO) log_info("Reading input partial variant tables and adding sample column") tables <- lapply( - snakemake@input, - function(path) { - read_tsv(path) %>% - mutate(SAMPLE = sub('\\.variants.tsv$', '', basename(path))) - } + snakemake@input, + function(path) { + read_tsv(path) %>% + mutate(SAMPLE = sub("\\.variants.tsv$", "", basename(path))) + } ) log_info("Binding and writing table") bind_rows(tables) %>% - write_tsv(snakemake@output$tsv) + write_tsv(snakemake@output$tsv) diff --git a/workflow/scripts/compile_vcf_fields_longer.R b/workflow/scripts/compile_vcf_fields_longer.R index 73dd243..7c5e7b3 100644 --- a/workflow/scripts/compile_vcf_fields_longer.R +++ b/workflow/scripts/compile_vcf_fields_longer.R @@ -5,19 +5,21 @@ log <- file(snakemake@log[[1]], open = "wt") sink(log, type = "message") sink(log, type = "output") -library(tidyverse) +library(readr) +library(dplyr) library(logger) + log_threshold(INFO) -log_info("Reading input partial tables with VCF extracted fields and adding sample column") +log_info("Reading input partial tables with VCF fields and adding sample column") tables <- lapply( - snakemake@input, - function(path) { - read_tsv(path) %>% - mutate(SAMPLE = sub('\\.vcf_fields.longer.tsv$', '', basename(path))) - } + snakemake@input, + function(path) { + read_tsv(path) %>% + mutate(SAMPLE = sub("\\.vcf_fields.longer.tsv$", "", basename(path))) + } ) log_info("Binding and writing table") bind_rows(tables) %>% - write_tsv(snakemake@output$tsv) + write_tsv(snakemake@output$tsv) diff --git a/workflow/scripts/dnds_data.R b/workflow/scripts/dnds_data.R index b71b23d..fc20d64 100644 --- a/workflow/scripts/dnds_data.R +++ b/workflow/scripts/dnds_data.R @@ -5,33 +5,36 @@ log <- file(snakemake@log[[1]], open = "wt") sink(log, type = "message") sink(log, type = "output") -library(tidyverse) +library(dplyr) +library(readr) +library(tidyr) library(logger) + log_threshold(INFO) # Read inputs log_info("Reading variants table") variants <- read_delim( - snakemake@input[["variants"]], - col_select = c( - "SAMPLE", - "VARIANT_NAME", - "REGION", - "ALT_FREQ", - "SYNONYMOUS", - "POS" - ) + snakemake@input[["variants"]], + col_select = c( + "SAMPLE", + "VARIANT_NAME", + "REGION", + "ALT_FREQ", + "SYNONYMOUS", + "POS" + ) ) log_info("Reading metadata table") metadata <- read_delim(snakemake@input[["metadata"]]) %>% - mutate( - interval = as.numeric( - as.Date(CollectionDate) - min(as.Date(CollectionDate)) - ) - ) %>% - select(ID, interval) %>% - rename(SAMPLE = ID) + mutate( + interval = as.numeric( + as.Date(CollectionDate) - min(as.Date(CollectionDate)) + ) + ) %>% + select(ID, interval) %>% + rename(SAMPLE = ID) log_debug("Adding metadata to variants table") variants <- left_join(variants, metadata) @@ -41,28 +44,28 @@ N_S_position <- read_delim(snakemake@input[["n_s_sites"]]) log_info("Computing dN/dS over time (NG86)") dn.ds <- variants %>% - group_by(SAMPLE, SYNONYMOUS) %>% - summarise( - Freq = sum(ALT_FREQ, na.rm = TRUE) - ) %>% - pivot_wider( - names_from = SYNONYMOUS, - values_from = Freq, - values_fill = 0 - ) %>% - transmute( - dn = No / sum(N_S_position$N), - ds = Yes / sum(N_S_position$S) - ) %>% - ungroup() %>% - left_join(unique(select(variants, SAMPLE, interval))) %>% - transmute( - sample = SAMPLE, - day = interval, - dN = dn, - dS = ds, - w = dn / ds - ) + group_by(SAMPLE, SYNONYMOUS) %>% + summarise( + Freq = sum(ALT_FREQ, na.rm = TRUE) + ) %>% + pivot_wider( + names_from = SYNONYMOUS, + values_from = Freq, + values_fill = 0 + ) %>% + transmute( + dn = No / sum(N_S_position$N), + ds = Yes / sum(N_S_position$S) + ) %>% + ungroup() %>% + left_join(unique(select(variants, SAMPLE, interval))) %>% + transmute( + sample = SAMPLE, + day = interval, + dN = dn, + dS = ds, + w = dn / ds + ) log_info("Writing results") write_csv(dn.ds, snakemake@output[["table"]]) diff --git a/workflow/scripts/download_context.R b/workflow/scripts/download_context.R index fefc7f6..8f0e119 100644 --- a/workflow/scripts/download_context.R +++ b/workflow/scripts/download_context.R @@ -6,9 +6,12 @@ sink(log, type = "message") sink(log, type = "output") library(GISAIDR) -library(tidyverse) +library(dplyr) +library(readr) library(glue) +library(lubridate) library(logger) + log_threshold(INFO) CHKPT.ERROR.MSG <- paste( @@ -34,9 +37,7 @@ needed.columns <- c( ) needed.columns.mask <- needed.columns %in% colnames(sample.metadata) if (!all(needed.columns.mask)) { - print(glue( - "Please ensure column '{needed.columns[!needed.columns.mask]}' is present" - )) + log_error("Please ensure column '{needed.columns[!needed.columns.mask]}' is present") stop(glue( "Missing columns in '{snakemake@input[['metadata']]}'. Alternatively:\n{CHKPT.ERROR.MSG}" )) @@ -51,24 +52,20 @@ log_info("Getting time windows for context samles") # Get time windows dates <- sample.metadata %>% pull(snakemake@params[["date_column"]]) %>% - as.numeric + as.numeric() window.quantile.offset <- (1 - snakemake@params[["date_window_span"]]) / 2 min.date <- as_date(quantile(dates, window.quantile.offset, na.rm = TRUE)) max.date <- as_date(quantile(dates, 1 - window.quantile.offset, na.rm = TRUE)) padded.min.date <- min.date - snakemake@params[["date_window_paddding_days"]] padded.max.date <- max.date + snakemake@params[["date_window_paddding_days"]] -print(glue( - "Time window (span={snakemake@params[['date_window_span']]}): {round(interval(min.date, max.date) / days(1))} days (from {min.date} to {max.date})" -)) -print(glue( - "Padded time window (padding={snakemake@params[['date_window_paddding_days']]} days): {round(interval(padded.min.date, padded.max.date) / days(1))} days (from {padded.min.date} to {padded.max.date})" -)) +log_info("Time window (span={snakemake@params[['date_window_span']]}): {round(interval(min.date, max.date) / days(1))} days (from {min.date} to {max.date})") +log_info("Padded time window (padding={snakemake@params[['date_window_paddding_days']]} days): {round(interval(padded.min.date, padded.max.date) / days(1))} days (from {padded.min.date} to {padded.max.date})") log_info("Getting locations for context samples") # Get locations (if there are multiple, sample from all of them) locations <- sample.metadata %>% pull(snakemake@params[["location_column"]]) %>% - unique + unique() # GISAID login creds.list <- yaml::read_yaml(snakemake@params[["gisaid_creds"]]) @@ -105,7 +102,7 @@ samples.accids <- sample.metadata %>% pull(snakemake@params[["samples_gisaid_accession_column"]]) filtered.accids <- metadata %>% filter(accession_id %in% samples.accids) metadata <- metadata %>% filter(!accession_id %in% samples.accids) -print(glue("{nrow(metadata)} accession_ids remaining after GISAID ID filter")) +log_info("{nrow(metadata)} accession_ids remaining after GISAID ID filter") # Checkpoint: enforce a minimum number of samples to have at least # as many possible combinations as random subsample replicates. diff --git a/workflow/scripts/filter_tsv.R b/workflow/scripts/filter_tsv.R index 69801e1..502ed78 100644 --- a/workflow/scripts/filter_tsv.R +++ b/workflow/scripts/filter_tsv.R @@ -5,8 +5,11 @@ log <- file(snakemake@log[[1]], open = "wt") sink(log, type = "message") sink(log, type = "output") -library(tidyverse) +library(dplyr) +library(readr) +library(stringr) library(logger) + log_threshold(INFO) # Read inputs diff --git a/workflow/scripts/format_vcf_fields_longer.R b/workflow/scripts/format_vcf_fields_longer.R index 407e9fb..a0a903e 100644 --- a/workflow/scripts/format_vcf_fields_longer.R +++ b/workflow/scripts/format_vcf_fields_longer.R @@ -5,62 +5,67 @@ log <- file(snakemake@log[[1]], open = "wt") sink(log, type = "message") sink(log, type = "output") -library(tidyverse) +library(readr) +library(dplyr) +library(tidyr) +library(stringr) +library(purrr) +library(rlang) +library(glue) +library(logger) empty.to.na <- function(x) { - x[x == ""] <- NA - x + x[x == ""] <- NA + x } # Replace "" values with NA in R filter list # Snakemake passes filters like: list(ERRORS = c("")) +log_info("Preprocessing data") filter.include <- lapply(snakemake@params$filter_include, empty.to.na) filter.exclude <- lapply(snakemake@params$filter_exclude, empty.to.na) # Process input table +log_info("Applying filters and writing results") read_tsv(snakemake@input$tsv) %>% - - # Separate -delimited "...[*]..." columns (e.g. ANN[*].EFFECT) - separate_rows( - contains("[*]"), - sep = snakemake@params$sep, - convert = TRUE - ) %>% - - # Rename "...[*]..." columns using the provided lookup via Snakemake config - rename(all_of(unlist(snakemake@params$colnames_mapping))) %>% - - # Separate &-delimited error column (more than one error/warning/info message per row is possible) - mutate(split_errors = strsplit(ERRORS, "&")) %>% - # Keep rows with none of the excluded ERRORS terms, if any - filter(map_lgl(split_errors, ~ !any(. %in% filter.exclude[["ERRORS"]]))) %>% - select(-split_errors) %>% - - # Apply dynamic filters from the Snakemake config: - # map2 pairs column names (.x) with value vectors (.y) and builds boolean expressions. - # Inside the expr call, !! injects a single value into each expression. - # The resulting list of expressions is spliced with !!! so each becomes its - # own condition as if written directly inside the filter call. - filter( - # Keep variants that include the required values in each defined field (e.g. empty ERRORS) - !!!map2( - names(filter.include), - filter.include, - ~ expr(.data[[!!.x]] %in% !!.y) - ), - # Keep variants that exclude the required values in each defined field (e.g. EFFECT != "upstream_gene_variant") - !!!map2( - names(filter.exclude), - filter.exclude, - ~ expr(!(.data[[!!.x]] %in% !!.y)) - ) - ) %>% - - # Keep unique rows - distinct() %>% - - # Assign variant name using the pattern defined via Snakemake config - mutate(VARIANT_NAME = str_glue(snakemake@params$variant_name_pattern)) %>% - - # Write output file - write_tsv(snakemake@output$tsv) + + # Separate -delimited "...[*]..." columns (e.g. ANN[*].EFFECT) + separate_rows( + contains("[*]"), + sep = snakemake@params$sep, + convert = TRUE + ) %>% + + # Rename "...[*]..." columns using the provided lookup via Snakemake config + rename(all_of(unlist(snakemake@params$colnames_mapping))) %>% + + # Separate &-delimited error column (more than one error/warning/info message per row is possible) + mutate(split_errors = strsplit(ERRORS, "&")) %>% + # Keep rows with none of the excluded ERRORS terms, if any + filter(map_lgl(split_errors, ~ !any(. %in% filter.exclude[["ERRORS"]]))) %>% + select(-split_errors) %>% + + # Apply filters + filter( + # Keep variants that include required values in each field + !!!map2( + names(filter.include), + filter.include, + ~ expr(.data[[!!.x]] %in% !!.y) + ), + # Keep variants that exclude required values in each field + !!!map2( + names(filter.exclude), + filter.exclude, + ~ expr(!(.data[[!!.x]] %in% !!.y)) + ) + ) %>% + + # Keep unique rows + distinct() %>% + + # Assign variant name using the pattern defined via Snakemake config + mutate(VARIANT_NAME = str_glue(snakemake@params$variant_name_pattern)) %>% + + # Write output file + write_tsv(snakemake@output$tsv) diff --git a/workflow/scripts/merge_annotation.R b/workflow/scripts/merge_annotation.R index fb9dcf5..0c21af6 100644 --- a/workflow/scripts/merge_annotation.R +++ b/workflow/scripts/merge_annotation.R @@ -5,8 +5,10 @@ log <- file(snakemake@log[[1]], open = "wt") sink(log, type = "message") sink(log, type = "output") -library(tidyverse) +library(dplyr) +library(readr) library(logger) + log_threshold(INFO) log_info("Reading variants table, replacing REGION with the reference name") diff --git a/workflow/scripts/report/af_time_correlation_data.R b/workflow/scripts/report/af_time_correlation_data.R index 6b6aafc..d3415c0 100644 --- a/workflow/scripts/report/af_time_correlation_data.R +++ b/workflow/scripts/report/af_time_correlation_data.R @@ -5,7 +5,10 @@ log <- file(snakemake@log[[1]], open = "wt") sink(log, type = "message") sink(log, type = "output") -library(tidyverse) +library(dplyr) +library(readr) +library(tidyr) +library(tibble) library(logger) log_threshold(INFO) diff --git a/workflow/scripts/report/af_time_correlation_plot.R b/workflow/scripts/report/af_time_correlation_plot.R index 9ee9a19..42f5b75 100644 --- a/workflow/scripts/report/af_time_correlation_plot.R +++ b/workflow/scripts/report/af_time_correlation_plot.R @@ -5,7 +5,9 @@ log <- file(snakemake@log[[1]], open = "wt") sink(log, type = "message") sink(log, type = "output") -library(tidyverse) +library(dplyr) +library(readr) +library(ggplot2) library(ggrepel) library(logger) log_threshold(INFO) diff --git a/workflow/scripts/report/af_trajectory_panel_plot.R b/workflow/scripts/report/af_trajectory_panel_plot.R index d8f1d62..61e7190 100644 --- a/workflow/scripts/report/af_trajectory_panel_plot.R +++ b/workflow/scripts/report/af_trajectory_panel_plot.R @@ -7,7 +7,11 @@ sink(log, type = "output") set.seed(snakemake@params$random_color_seed) # seed for sampling colors -library(tidyverse) +library(dplyr) +library(readr) +library(ggplot2) +library(glue) +library(logger) library(logger) log_threshold(INFO) @@ -40,7 +44,7 @@ p <- variants %>% aes( x = interval, y = ALT_FREQ, - color = reorder(glue::glue("{gPOS}\n{VARIANT_NAME}"), POS) + color = reorder(glue("{gPOS}\n{VARIANT_NAME}"), POS) ) + scale_color_manual(values = selected.colors) + geom_point() + diff --git a/workflow/scripts/report/allele_freq_tree_data.R b/workflow/scripts/report/allele_freq_tree_data.R index f7bd7aa..7272bfa 100644 --- a/workflow/scripts/report/allele_freq_tree_data.R +++ b/workflow/scripts/report/allele_freq_tree_data.R @@ -5,7 +5,9 @@ log <- file(snakemake@log[[1]], open = "wt") sink(log, type = "message") sink(log, type = "output") -library(tidyverse) +library(dplyr) +library(readr) +library(tibble) library(data.table) library(ape) library(logger) diff --git a/workflow/scripts/report/allele_freq_tree_plot.R b/workflow/scripts/report/allele_freq_tree_plot.R index 5ccb603..b25404f 100644 --- a/workflow/scripts/report/allele_freq_tree_plot.R +++ b/workflow/scripts/report/allele_freq_tree_plot.R @@ -5,8 +5,10 @@ log <- file(snakemake@log[[1]], open = "wt") sink(log, type = "message") sink(log, type = "output") -library(tidyverse) +library(dplyr) +library(readr) library(ape) +library(ggplot2) library(ggtree) library(logger) diff --git a/workflow/scripts/report/context_phylogeny_data.R b/workflow/scripts/report/context_phylogeny_data.R index 704df80..eb4348a 100644 --- a/workflow/scripts/report/context_phylogeny_data.R +++ b/workflow/scripts/report/context_phylogeny_data.R @@ -5,7 +5,9 @@ log <- file(snakemake@log[[1]], open = "wt") sink(log, type = "message") sink(log, type = "output") -library(tidyverse) +library(dplyr) +library(readr) +library(tibble) library(jsonlite) library(ape) library(logger) diff --git a/workflow/scripts/report/context_phylogeny_plot.R b/workflow/scripts/report/context_phylogeny_plot.R index 8b56164..2aa0781 100644 --- a/workflow/scripts/report/context_phylogeny_plot.R +++ b/workflow/scripts/report/context_phylogeny_plot.R @@ -5,9 +5,11 @@ log <- file(snakemake@log[[1]], open = "wt") sink(log, type = "message") sink(log, type = "output") -library(tidyverse) +library(dplyr) +library(readr) library(jsonlite) library(ape) +library(ggplot2) library(ggtree) library(logger) diff --git a/workflow/scripts/report/demix_plot.R b/workflow/scripts/report/demix_plot.R index fc49544..1cf1c3d 100644 --- a/workflow/scripts/report/demix_plot.R +++ b/workflow/scripts/report/demix_plot.R @@ -5,8 +5,11 @@ log <- file(snakemake@log[[1]], open = "wt") sink(log, type = "message") sink(log, type = "output") -library(tidyverse) +library(dplyr) +library(readr) +library(ggplot2) library(logger) + log_threshold(INFO) # Import file with plots style diff --git a/workflow/scripts/report/demix_plot_data.R b/workflow/scripts/report/demix_plot_data.R index 2aafcde..cb0c08b 100644 --- a/workflow/scripts/report/demix_plot_data.R +++ b/workflow/scripts/report/demix_plot_data.R @@ -5,8 +5,10 @@ log <- file(snakemake@log[[1]], open = "wt") sink(log, type = "message") sink(log, type = "output") -library(tidyverse) +library(dplyr) +library(readr) library(logger) + log_threshold(INFO) # Read inputs diff --git a/workflow/scripts/report/diversity_data.R b/workflow/scripts/report/diversity_data.R index d321bde..9ca5918 100644 --- a/workflow/scripts/report/diversity_data.R +++ b/workflow/scripts/report/diversity_data.R @@ -9,10 +9,12 @@ set.seed(snakemake@params$seed) library(ape) library(pegas) +library(future) library(future.apply) -library(tidyverse) +library(readr) library(jsonlite) library(logger) + log_threshold(INFO) # Pi calculation diff --git a/workflow/scripts/report/diversity_plot.R b/workflow/scripts/report/diversity_plot.R index b6c4c9c..7118029 100644 --- a/workflow/scripts/report/diversity_plot.R +++ b/workflow/scripts/report/diversity_plot.R @@ -5,9 +5,12 @@ log <- file(snakemake@log[[1]], open = "wt") sink(log, type = "message") sink(log, type = "output") -library(tidyverse) +library(dplyr) +library(readr) +library(ggplot2) library(jsonlite) library(logger) + log_threshold(INFO) # Import file with plots style diff --git a/workflow/scripts/report/dnds_plots.R b/workflow/scripts/report/dnds_plots.R index ae44d98..62b2e41 100644 --- a/workflow/scripts/report/dnds_plots.R +++ b/workflow/scripts/report/dnds_plots.R @@ -5,8 +5,12 @@ log <- file(snakemake@log[[1]], open = "wt") sink(log, type = "message") sink(log, type = "output") -library(tidyverse) +library(dplyr) +library(readr) +library(tidyr) +library(ggplot2) library(logger) + log_threshold(INFO) # Import file with plots style diff --git a/workflow/scripts/report/heatmap.R b/workflow/scripts/report/heatmap.R index 5955a75..57952e4 100644 --- a/workflow/scripts/report/heatmap.R +++ b/workflow/scripts/report/heatmap.R @@ -5,8 +5,13 @@ log <- file(snakemake@log[[1]], open = "wt") sink(log, type = "message") sink(log, type = "output") -library(tidyverse) +library(dplyr) +library(readr) +library(tidyr) +library(tibble) +library(stringr) library(logger) + log_threshold(INFO) vcf <- read_tsv(snakemake@input[["vcf"]]) @@ -18,8 +23,7 @@ date_order <- read_csv(snakemake@input[["metadata"]]) %>% unique() # Create SNP variable and select useful variables from vcf -vcf <- vcf %>% - dplyr::select(VARIANT_NAME, SAMPLE, ALT_FREQ) +vcf <- vcf %>% select(VARIANT_NAME, SAMPLE, ALT_FREQ) vcf <- vcf %>% pivot_wider( diff --git a/workflow/scripts/report/nv_panel_data.R b/workflow/scripts/report/nv_panel_data.R index f49260f..5cb6880 100644 --- a/workflow/scripts/report/nv_panel_data.R +++ b/workflow/scripts/report/nv_panel_data.R @@ -5,9 +5,13 @@ log <- file(snakemake@log[[1]], open = "wt") sink(log, type = "message") sink(log, type = "output") -library(tidyverse) +library(dplyr) +library(readr) +library(stringr) +library(tibble) library(jsonlite) library(logger) + log_threshold(INFO) log_info("Reading variants") diff --git a/workflow/scripts/report/nv_panel_plot.R b/workflow/scripts/report/nv_panel_plot.R index 07cc1b0..da7eca7 100644 --- a/workflow/scripts/report/nv_panel_plot.R +++ b/workflow/scripts/report/nv_panel_plot.R @@ -5,10 +5,15 @@ log <- file(snakemake@log[[1]], open = "wt") sink(log, type = "message") sink(log, type = "output") -library(tidyverse) +library(dplyr) +library(readr) +library(ggplot2) library(ggpubr) library(jsonlite) +library(tibble) +library(purrr) library(logger) + log_threshold(INFO) # Import file with plots style diff --git a/workflow/scripts/report/polymorphic_sites_over_time_data.R b/workflow/scripts/report/polymorphic_sites_over_time_data.R index bf328f6..1100587 100644 --- a/workflow/scripts/report/polymorphic_sites_over_time_data.R +++ b/workflow/scripts/report/polymorphic_sites_over_time_data.R @@ -5,9 +5,11 @@ log <- file(snakemake@log[[1]], open = "wt") sink(log, type = "message") sink(log, type = "output") -library(tidyverse) +library(readr) +library(dplyr) library(jsonlite) library(logger) + log_threshold(INFO) log_info("Reading variants") diff --git a/workflow/scripts/report/polymorphic_sites_over_time_plot.R b/workflow/scripts/report/polymorphic_sites_over_time_plot.R index 83c1425..2596195 100644 --- a/workflow/scripts/report/polymorphic_sites_over_time_plot.R +++ b/workflow/scripts/report/polymorphic_sites_over_time_plot.R @@ -5,8 +5,11 @@ log <- file(snakemake@log[[1]], open = "wt") sink(log, type = "message") sink(log, type = "output") -library(tidyverse) +library(readr) +library(dplyr) +library(ggplot2) library(logger) + log_threshold(INFO) # Import file with plots style diff --git a/workflow/scripts/report/summary_table.R b/workflow/scripts/report/summary_table.R index b78a103..0f96f7d 100644 --- a/workflow/scripts/report/summary_table.R +++ b/workflow/scripts/report/summary_table.R @@ -5,8 +5,9 @@ log <- file(snakemake@log[[1]], open = "wt") sink(log, type = "message") sink(log, type = "output") -library(tidyverse) +library(dplyr) library(logger) + log_threshold(INFO) metadata <- read.csv(snakemake@input[["metadata"]]) diff --git a/workflow/scripts/report/time_signal_data.R b/workflow/scripts/report/time_signal_data.R index b270ebf..103e95b 100644 --- a/workflow/scripts/report/time_signal_data.R +++ b/workflow/scripts/report/time_signal_data.R @@ -5,7 +5,9 @@ log <- file(snakemake@log[[1]], open = "wt") sink(log, type = "message") sink(log, type = "output") -library(tidyverse) +library(readr) +library(dplyr) +library(tibble) library(jsonlite) library(ape) library(adephylo) diff --git a/workflow/scripts/report/time_signal_plot.R b/workflow/scripts/report/time_signal_plot.R index 27741f0..d9af70c 100644 --- a/workflow/scripts/report/time_signal_plot.R +++ b/workflow/scripts/report/time_signal_plot.R @@ -5,7 +5,9 @@ log <- file(snakemake@log[[1]], open = "wt") sink(log, type = "message") sink(log, type = "output") -library(tidyverse) +library(readr) +library(dplyr) +library(ggplot2) library(logger) log_threshold(INFO) diff --git a/workflow/scripts/summarise_demix.R b/workflow/scripts/summarise_demix.R index 895354f..9792202 100644 --- a/workflow/scripts/summarise_demix.R +++ b/workflow/scripts/summarise_demix.R @@ -5,8 +5,12 @@ log <- file(snakemake@log[[1]], open = "wt") sink(log, type = "message") sink(log, type = "output") -library(tidyverse) +library(dplyr) +library(readr) +library(tidyr) +library(stringr) library(logger) + log_threshold(INFO) # Empty dataframe to be filled with data From 9efd953d9d30d5c90c36c7a5323d0af9785fe285 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 4 Nov 2025 15:45:43 +0100 Subject: [PATCH 109/138] Update quarto call --- workflow/rules/report.smk | 5 ++--- 1 file changed, 2 insertions(+), 3 deletions(-) diff --git a/workflow/rules/report.smk b/workflow/rules/report.smk index 241711a..3a42acb 100644 --- a/workflow/rules/report.smk +++ b/workflow/rules/report.smk @@ -423,7 +423,7 @@ rule report: shadow: "shallow" input: qmd = Path(config["REPORT_QMD"]).resolve(), - demix = report(REPORT_DIR_PLOTS/"demix.png"), + demix = report(REPORT_DIR_PLOTS/"demix.png"), tree_ml = report(REPORT_DIR_PLOTS/"context_phylogeny.png"), diversity = report(REPORT_DIR_PLOTS/"diversity.png"), fig_cor = report(REPORT_DIR_PLOTS/"polymorphic_sites_over_time.png"), @@ -456,8 +456,7 @@ rule report: LOGDIR / "report" / "log.txt" shell: "set +o pipefail; " - "Rscript -e 'library(quarto)' " - "-e \"quarto_render(" + "Rscript -e \"quarto::quarto_render(" "input = '{input.qmd}', " "execute_params=list(" "ufboot_reps='{params.ufboot_reps}', " From 34f15b60a27bda3e223b9fa201329f58a98feefa Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 4 Nov 2025 15:45:57 +0100 Subject: [PATCH 110/138] Update quarto environment --- workflow/envs/quarto_render.yaml | 14 ++++++-------- 1 file changed, 6 insertions(+), 8 deletions(-) diff --git a/workflow/envs/quarto_render.yaml b/workflow/envs/quarto_render.yaml index 71fa4cb..3df14d2 100644 --- a/workflow/envs/quarto_render.yaml +++ b/workflow/envs/quarto_render.yaml @@ -1,12 +1,10 @@ channels: - conda-forge - - bioconda dependencies: - - r-base==4.3.1 - - r-gt==0.9.0 - - quarto==1.3.450 - - r-jsonlite==1.8.5 + - r-base==4.5.2 + - r-gt==1.1.0 + - quarto==1.8.25 + - deno==2.3.1 - r-tidyverse==2.0.0 - - r-quarto==1.2 - - r-heatmaply==1.4.2 - - r-readr==2.1.4 + - r-quarto==1.5.1 + - r-heatmaply==1.6.0 From d6dc8d1e014b9b938a07b53a8962adfa7c04620a Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 4 Nov 2025 16:31:36 +0100 Subject: [PATCH 111/138] Update CSS and add as report input --- config/README.md | 4 +--- config/config.yaml | 2 ++ config/report.styles.css | 49 ++++++++++++++++++++++----------------- template.qmd | 22 ++++++++---------- workflow/rules/report.smk | 4 +++- 5 files changed, 44 insertions(+), 37 deletions(-) diff --git a/config/README.md b/config/README.md index 04a94cb..d55d3e9 100644 --- a/config/README.md +++ b/config/README.md @@ -182,9 +182,7 @@ All of the following variables are pre-defined in [config.yaml](/config/config.y - `PLOTS`: path of the R script that sets the design and style of data visualizations. - `PLOT_GENOME_REGIONS`: path of a CSV file containing genome regions, e.g. SARS-CoV-2 non-structural protein (NSP) coordinates, for data visualization (columns: `region`, `start`, `end`). - `REPORT_QMD`: path of the report template in Quarto markdown (QMD) format. - -TODO: describe new entries in config.yaml -TODO: describe issues with many cores (rules freyja and snps_to_ancestor) +- `REPORT_CSS`: path of the report stylesheet definition in CSS format. ## Workflow graphs diff --git a/config/config.yaml b/config/config.yaml index c00cc02..c9e0373 100644 --- a/config/config.yaml +++ b/config/config.yaml @@ -97,3 +97,5 @@ PLOT_GENOME_REGIONS: "config/nsp_annotation.csv" REPORT_QMD: "template.qmd" +REPORT_CSS: + "config/report.styles.css" diff --git a/config/report.styles.css b/config/report.styles.css index 6282f9a..9ea2783 100644 --- a/config/report.styles.css +++ b/config/report.styles.css @@ -1,35 +1,42 @@ -.quarto-title-block .quarto-title-banner { - background: #27445C; - background-image: url(logo_pathogenomics.png); - background-size: 100px; - background-position: right; - background-repeat: no-repeat; - padding-right: 10px; - background-origin: content-box; - } - -.title { - font-family: "Noto Sans"; - color: #FFFFFF -} -.quarto-title p { - font-family: "Noto Sans"; - color: #FFFFFF -} html { font-family: "Noto Sans"; - color: #000000; text-align: justify; } +body { + color: black; +} + +.quarto-title-block .quarto-title-banner { + background-color: #27445C; + background-image: url("logo_pathogenomics.png"); + background-size: 100px; + background-position: right; + background-repeat: no-repeat; + background-origin: content-box; + padding-right: 10px; + background-blend-mode: normal; + filter: none; + mix-blend-mode: normal; + color: white; +} +.quarto-title-block .quarto-title-banner .title, +.quarto-title-block .quarto-title-banner p { + color: inherit; + -webkit-text-fill-color: inherit; + opacity: 1; +} + h1, h2, h3 { text-align: left; font-weight: bold; + color: #27445C } + #quarto-sidebar-toc-left { text-align: left; } .header-section-number { - color:#000000 -} \ No newline at end of file + color: #27445C +} diff --git a/template.qmd b/template.qmd index d0772bc..c6aba13 100644 --- a/template.qmd +++ b/template.qmd @@ -15,9 +15,10 @@ format: toc-location: left toc-title: Summary number-sections: true -css: config/report.styles.css +css: "`r params$css`" editor: source params: + css: "" ufboot_reps: "" shalrt_reps: "" min_ivar_freq: "" @@ -84,9 +85,9 @@ table <- read.csv(params$table) n.samples <- table %>% pull(Sample) %>% unique() %>% length() # Heatmap -heatmap.df <- read_csv(params$heat_tab) +heatmap.df <- read.csv(params$heat_tab) row.names(heatmap.df) <- heatmap.df$`...1` -heatmap.df <- heatmap.df[-1] +heatmap.df[1] <- NULL cor.mat <- cor(heatmap.df, method = params$cor_method) cor.mat[is.na(cor.mat)] <- 0 if (params$cor_method == "pearson") { @@ -114,8 +115,7 @@ freyja.timestamp <- read_file(params$freyja_ts) %>% ## Summary of the target samples dataset -```{r summary_table, echo = F,message = F} -#| label: tbl-summary +```{r tbl-summary, echo = F,message = F} #| tbl-cap: Target dataset summary table %>% @@ -268,19 +268,17 @@ line represents the linear model fit.](`r params$tempest`){#fig-tempest} ### Correlation between alternative alleles To detect possible interactions between mutations, pairwise correlation between allele -frequencies are calculated (@fig-heatmap). The heatmap is an interactive figure that allows +frequencies have been calculated (@fig-heatmap). The heatmap is an interactive figure that allows zooming in on specific regions. -```{r heatmap, echo = F, message = F, fig.align = 'center'} -#| label: fig-heatmap -#| fig-width: 6.27 -#| fig-height: 6.27 +```{r fig-heatmap, echo = F, message = F, warning = F, fig.align = 'center'} #| fig-cap: "Interactive hierarchically clustered heatmap of the pairwisecorrelation coefficients between the time series of allele frequencies in the case study." heatmaply_cor( cor.mat, - fontsize_row = 8, - fontsize_col = 8, + grid_gap = 0.25, + fontsize_row = 7, + fontsize_col = 7, column_text_angle = 45, width = 600, height = 600 diff --git a/workflow/rules/report.smk b/workflow/rules/report.smk index 3a42acb..54908eb 100644 --- a/workflow/rules/report.smk +++ b/workflow/rules/report.smk @@ -423,6 +423,7 @@ rule report: shadow: "shallow" input: qmd = Path(config["REPORT_QMD"]).resolve(), + css = Path(config["REPORT_CSS"]).resolve(), demix = report(REPORT_DIR_PLOTS/"demix.png"), tree_ml = report(REPORT_DIR_PLOTS/"context_phylogeny.png"), diversity = report(REPORT_DIR_PLOTS/"diversity.png"), @@ -457,8 +458,9 @@ rule report: shell: "set +o pipefail; " "Rscript -e \"quarto::quarto_render(" - "input = '{input.qmd}', " + "input = '{input.qmd:q}', " "execute_params=list(" + "css='{input.css:q}', " "ufboot_reps='{params.ufboot_reps}', " "shalrt_reps='{params.shalrt_reps}', " "min_ivar_freq='{params.min_ivar_freq}', " From 2684921075a36a287a69453ad6d04a0bf5b37a01 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 4 Nov 2025 16:48:24 +0100 Subject: [PATCH 112/138] Update Freyja version --- workflow/envs/freyja.yaml | 2 +- workflow/rules/demix.smk | 7 +------ 2 files changed, 2 insertions(+), 7 deletions(-) diff --git a/workflow/envs/freyja.yaml b/workflow/envs/freyja.yaml index ff61e67..6673ab1 100644 --- a/workflow/envs/freyja.yaml +++ b/workflow/envs/freyja.yaml @@ -2,4 +2,4 @@ channels: - conda-forge - bioconda dependencies: - - freyja==2.0.0 + - freyja==2.0.1 diff --git a/workflow/rules/demix.smk b/workflow/rules/demix.smk index c16cc3e..367e7b1 100644 --- a/workflow/rules/demix.smk +++ b/workflow/rules/demix.smk @@ -67,12 +67,6 @@ rule demix: log: LOGDIR / "demix" / "{sample}.log.txt" shell: - "RAYON_NUM_THREADS={threads} " - "JULIA_NUM_THREADS={threads} " - "BLAS_NUM_THREADS={threads} " - "OPENBLAS_NUM_THREADS={threads}" - "MKL_NUM_THREADS={threads} " - "OMP_NUM_THREADS={threads} " "freyja demix " "{input.variants_file:q} " "{input.depth_file:q} " @@ -88,6 +82,7 @@ rule demix: "--relaxedthresh {params.relaxed_mrca_thresh} " "--solver {params.solver} " "--output {output.demix_file} " + "--max-solver-threads {threads} " ">{log} 2>&1" From d9bb2cef416cc3a36e7282ccc87fa9ac0b1cfecb Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 4 Nov 2025 17:19:35 +0100 Subject: [PATCH 113/138] Update table concatenation Simplify TSV concatenation and reuse a rule. This avoids problems and allows reusing the rule for any other set of tables. --- workflow/rules/vaf.smk | 41 ++++++++++---------- workflow/scripts/compile_variants.R | 25 ------------ workflow/scripts/compile_vcf_fields_longer.R | 25 ------------ workflow/scripts/format_vcf_fields_longer.R | 8 +++- workflow/scripts/merge_annotation.R | 5 ++- 5 files changed, 31 insertions(+), 73 deletions(-) delete mode 100644 workflow/scripts/compile_variants.R delete mode 100644 workflow/scripts/compile_vcf_fields_longer.R diff --git a/workflow/rules/vaf.smk b/workflow/rules/vaf.smk index 6253941..ea5c7b9 100644 --- a/workflow/rules/vaf.smk +++ b/workflow/rules/vaf.smk @@ -149,37 +149,43 @@ rule extract_vcf_fields: rule format_vcf_fields_longer: conda: "../envs/renv.yaml" params: + sample = "{sample}", colnames_mapping = config["ANNOTATION"]["SNPEFF_COLS"], filter_include = config["ANNOTATION"]["FILTER_INCLUDE"], filter_exclude = config["ANNOTATION"]["FILTER_EXCLUDE"], variant_name_pattern = lambda wildcards: config["ANNOTATION"]["VARIANT_NAME_PATTERN"], # lambda to deactivate automatic wildcard expansion in pattern sep = ",", input: - tsv = OUTDIR/"vaf"/"{sample}.vcf_fields.tsv" + tsv = OUTDIR/"vaf"/"{sample}.vcf_fields.tsv", output: - tsv = OUTDIR/"vaf"/"{sample}.vcf_fields.longer.tsv" + tsv = OUTDIR/"vaf"/"{sample}.vcf_fields.longer.tsv", log: LOGDIR / "format_vcf_fields_longer" / "{sample}.log.txt" script: "../scripts/format_vcf_fields_longer.R" -rule compile_vcf_fields_longer: - threads: 1 - conda: "../envs/renv.yaml" - input: expand(OUTDIR/"vaf"/"{sample}.vcf_fields.longer.tsv", sample=iter_samples()) +rule concat_vcf_fields: + params: + sep = "\t", + input: + expand(OUTDIR/"vaf"/"{sample}.vcf_fields.longer.tsv", sample=iter_samples()), output: - tsv = OUTDIR/f"{OUTPUT_NAME}.vcf_fields.longer.tsv" - log: - LOGDIR / "compile_vcf_fields_longer" / "log.txt" - script: - "../scripts/compile_vcf_fields_longer.R" + OUTDIR/f"{OUTPUT_NAME}.vcf_fields.longer.tsv", + run: + import pandas as pd + from functools import reduce + reduce( + lambda a, b: pd.concat((a, b), axis="rows", ignore_index=True), + (pd.read_csv(path, sep=params.sep) for path in input) + ).to_csv(output[0], sep=params.sep, index=False) rule merge_annotation: threads: 1 conda: "../envs/renv.yaml" params: + sample = "{sample}", ref_name = config["ALIGNMENT_REFERENCE"], input: tsv = OUTDIR/"vaf"/"{sample}.masked.prefiltered.tsv", @@ -192,13 +198,8 @@ rule merge_annotation: "../scripts/merge_annotation.R" -rule compile_variants: - threads: 1 - conda: "../envs/renv.yaml" - input: expand(OUTDIR/"vaf"/"{sample}.variants.tsv", sample=iter_samples()) +use rule concat_vcf_fields as concat_variants with: + input: + expand(OUTDIR/"vaf"/"{sample}.variants.tsv", sample=iter_samples()), output: - tsv = OUTDIR/f"{OUTPUT_NAME}.variants.tsv" - log: - LOGDIR / "compile_variants" / "log.txt" - script: - "../scripts/compile_variants.R" + OUTDIR/f"{OUTPUT_NAME}.variants.tsv", diff --git a/workflow/scripts/compile_variants.R b/workflow/scripts/compile_variants.R deleted file mode 100644 index a3f435a..0000000 --- a/workflow/scripts/compile_variants.R +++ /dev/null @@ -1,25 +0,0 @@ -#!/usr/bin/env Rscript - -# Write stdout and stderr to log file -log <- file(snakemake@log[[1]], open = "wt") -sink(log, type = "message") -sink(log, type = "output") - -library(readr) -library(dplyr) -library(logger) - -log_threshold(INFO) - -log_info("Reading input partial variant tables and adding sample column") -tables <- lapply( - snakemake@input, - function(path) { - read_tsv(path) %>% - mutate(SAMPLE = sub("\\.variants.tsv$", "", basename(path))) - } -) - -log_info("Binding and writing table") -bind_rows(tables) %>% - write_tsv(snakemake@output$tsv) diff --git a/workflow/scripts/compile_vcf_fields_longer.R b/workflow/scripts/compile_vcf_fields_longer.R deleted file mode 100644 index 7c5e7b3..0000000 --- a/workflow/scripts/compile_vcf_fields_longer.R +++ /dev/null @@ -1,25 +0,0 @@ -#!/usr/bin/env Rscript - -# Write stdout and stderr to log file -log <- file(snakemake@log[[1]], open = "wt") -sink(log, type = "message") -sink(log, type = "output") - -library(readr) -library(dplyr) -library(logger) - -log_threshold(INFO) - -log_info("Reading input partial tables with VCF fields and adding sample column") -tables <- lapply( - snakemake@input, - function(path) { - read_tsv(path) %>% - mutate(SAMPLE = sub("\\.vcf_fields.longer.tsv$", "", basename(path))) - } -) - -log_info("Binding and writing table") -bind_rows(tables) %>% - write_tsv(snakemake@output$tsv) diff --git a/workflow/scripts/format_vcf_fields_longer.R b/workflow/scripts/format_vcf_fields_longer.R index a0a903e..26c5512 100644 --- a/workflow/scripts/format_vcf_fields_longer.R +++ b/workflow/scripts/format_vcf_fields_longer.R @@ -64,8 +64,12 @@ read_tsv(snakemake@input$tsv) %>% # Keep unique rows distinct() %>% - # Assign variant name using the pattern defined via Snakemake config - mutate(VARIANT_NAME = str_glue(snakemake@params$variant_name_pattern)) %>% + mutate( + # Assign variant name using the pattern defined via Snakemake config + VARIANT_NAME = str_glue(snakemake@params$variant_name_pattern), + # Assign sample name + SAMPLE = snakemake@params$sample + ) %>% # Write output file write_tsv(snakemake@output$tsv) diff --git a/workflow/scripts/merge_annotation.R b/workflow/scripts/merge_annotation.R index 0c21af6..9c322c4 100644 --- a/workflow/scripts/merge_annotation.R +++ b/workflow/scripts/merge_annotation.R @@ -72,7 +72,10 @@ merged <- left_join( "REF_VCF" = "REF", "ALT_VCF" = "ALT" ) -) +) %>% + mutate( + SAMPLE = snakemake@params$sample + ) log_info("Saving results") write_tsv( From 13ec31be708e3134a8219a9961fb4f16d8974e00 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 4 Nov 2025 17:23:04 +0100 Subject: [PATCH 114/138] Update N/S rules naming --- config/README.md | 2 +- workflow/rules/evolution.smk | 10 +++++----- workflow/scripts/dnds_data.R | 6 +++--- ...N_S_sites_from_fasta.py => n_s_sites_from_fasta.py} | 0 4 files changed, 9 insertions(+), 9 deletions(-) rename workflow/scripts/{N_S_sites_from_fasta.py => n_s_sites_from_fasta.py} (100%) diff --git a/config/README.md b/config/README.md index d55d3e9..7ffdeca 100644 --- a/config/README.md +++ b/config/README.md @@ -157,7 +157,7 @@ All of the following variables are pre-defined in [config.yaml](/config/config.y - `WIDTH`: number of sites within windows. - `STEP`: number of sites between windows. - `GB_FEATURES`: optional mapping to filter which features from the GenBank file are used - by some analyses (e.g. rules `window` and `N_S_sites`). + by some analyses (e.g. rules `window` and `n_s_sites`). If `GB_FEATURES` is empty or unset, all features are used. Filtering is applied in order: - `INCLUDE`: mapping of qualifier names to sequences of values. diff --git a/workflow/rules/evolution.smk b/workflow/rules/evolution.smk index 2951763..ee7156d 100644 --- a/workflow/rules/evolution.smk +++ b/workflow/rules/evolution.smk @@ -14,7 +14,7 @@ rule filter_genbank_features: "../scripts/filter_genbank_features.py" -rule N_S_sites: +rule n_s_sites: threads: 1 conda: "../envs/biopython.yaml" params: @@ -24,17 +24,17 @@ rule N_S_sites: gb = OUTDIR/"reference.cds.gb", genetic_code = Path(config["GENETIC_CODE_JSON"]).resolve(), output: - csv = temp(OUTDIR/f"{OUTPUT_NAME}.ancestor.N_S.sites.csv"), + csv = temp(OUTDIR/f"{OUTPUT_NAME}.ancestor.n_s.sites.csv"), log: - LOGDIR / "N_S_sites" / "log.txt" + LOGDIR / "n_s_sites" / "log.txt" script: - "../scripts/N_S_sites_from_fasta.py" + "../scripts/n_s_sites_from_fasta.py" rule dnds_data: conda: "../envs/renv.yaml" input: - n_s_sites = OUTDIR/f"{OUTPUT_NAME}.ancestor.N_S.sites.csv", + n_s_sites = OUTDIR/f"{OUTPUT_NAME}.ancestor.n_s.sites.csv", variants = OUTDIR/f"{OUTPUT_NAME}.variants.tsv", metadata = config["METADATA"] output: diff --git a/workflow/scripts/dnds_data.R b/workflow/scripts/dnds_data.R index fc20d64..f545c27 100644 --- a/workflow/scripts/dnds_data.R +++ b/workflow/scripts/dnds_data.R @@ -40,7 +40,7 @@ log_debug("Adding metadata to variants table") variants <- left_join(variants, metadata) log_info("Reading N/S sites") -N_S_position <- read_delim(snakemake@input[["n_s_sites"]]) +n_s_position <- read_delim(snakemake@input[["n_s_sites"]]) log_info("Computing dN/dS over time (NG86)") dn.ds <- variants %>% @@ -54,8 +54,8 @@ dn.ds <- variants %>% values_fill = 0 ) %>% transmute( - dn = No / sum(N_S_position$N), - ds = Yes / sum(N_S_position$S) + dn = No / sum(n_s_position$N), + ds = Yes / sum(n_s_position$S) ) %>% ungroup() %>% left_join(unique(select(variants, SAMPLE, interval))) %>% diff --git a/workflow/scripts/N_S_sites_from_fasta.py b/workflow/scripts/n_s_sites_from_fasta.py similarity index 100% rename from workflow/scripts/N_S_sites_from_fasta.py rename to workflow/scripts/n_s_sites_from_fasta.py From a97a251c04351b6d2da7ea2b375d5642ec509066 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 4 Nov 2025 17:42:07 +0100 Subject: [PATCH 115/138] Improve input description and instructions --- config/README.md | 12 +++++++----- 1 file changed, 7 insertions(+), 5 deletions(-) diff --git a/config/README.md b/config/README.md index 7ffdeca..14fa5aa 100644 --- a/config/README.md +++ b/config/README.md @@ -16,12 +16,14 @@ Instead, use `--software-deployment-method conda`. The workflow requires a set of FASTA files (one per target sample), a corresponding set of BAM files (also one per target sample), and a metadata table in CSV format with one row per -sample. The metadata must include the following columns: unique sample identifier (default column `ID`, -used to match sequencing files with metadata), the date the sample was collected (default `CollectionDate`), -the location where the sample was collected (default `ResidenceCity`), and GISAID accession (default `GISAIDEPI`). -The default column names but can be customized if needed via the workflow parameters. +sample. The metadata must include at least the following columns: -These parameters are set in two configuration files in YAML format: +- `ID`: unique sample identifier, used to match sequencing files with metadata. +- `CollectionDate`: the date the sample was collected (YYYY-MM-DD). +- `ResidenceCity`: the location where the sample was collected. +- `GISAIDEPI`: GISAID accession identifier (`EPI_ISL_...` or empty). + +The path to these input files is set in two configuration files in YAML format: [config.yaml](/config/config.yaml) (for general workflow settings) and [targets.yaml](/config/targets.yaml) (for specific dataset-related settings). The latter must be modified by the user to point the `SAMPLES` and `METADATA` From b5aa082de6a599febcd4fa207164adcd260e2fb3 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 4 Nov 2025 17:48:04 +0100 Subject: [PATCH 116/138] Fix naming of variants table --- workflow/rules/report.smk | 2 +- workflow/scripts/report/heatmap.R | 10 +++++----- 2 files changed, 6 insertions(+), 6 deletions(-) diff --git a/workflow/rules/report.smk b/workflow/rules/report.smk index 54908eb..c8ee402 100644 --- a/workflow/rules/report.smk +++ b/workflow/rules/report.smk @@ -30,7 +30,7 @@ rule demix_plot: rule heatmap_plot_data: conda: "../envs/renv.yaml" input: - vcf = OUTDIR/f"{OUTPUT_NAME}.variants.tsv", + variants = OUTDIR/f"{OUTPUT_NAME}.variants.tsv", metadata = config["METADATA"] output: table = report(REPORT_DIR_TABLES/"heatmap.csv") diff --git a/workflow/scripts/report/heatmap.R b/workflow/scripts/report/heatmap.R index 57952e4..27a8dfc 100644 --- a/workflow/scripts/report/heatmap.R +++ b/workflow/scripts/report/heatmap.R @@ -14,7 +14,7 @@ library(logger) log_threshold(INFO) -vcf <- read_tsv(snakemake@input[["vcf"]]) +variants <- read_tsv(snakemake@input[["variants"]]) # Obtain sample names ordered by CollectionDate date_order <- read_csv(snakemake@input[["metadata"]]) %>% @@ -22,10 +22,10 @@ date_order <- read_csv(snakemake@input[["metadata"]]) %>% pull(ID) %>% unique() -# Create SNP variable and select useful variables from vcf -vcf <- vcf %>% select(VARIANT_NAME, SAMPLE, ALT_FREQ) +# Create SNP variable and select useful variables +variants <- variants %>% select(VARIANT_NAME, SAMPLE, ALT_FREQ) -vcf <- vcf %>% +variants <- variants %>% pivot_wider( names_from = VARIANT_NAME, values_from = ALT_FREQ, @@ -38,4 +38,4 @@ vcf <- vcf %>% column_to_rownames(var = "SAMPLE") log_info("Saving table to create heatmap") -write.csv(vcf, snakemake@output[["table"]]) +write.csv(variants, snakemake@output[["table"]]) From 969544a599eefe834e9eaf99842c9593bebe0122 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 4 Nov 2025 18:07:23 +0100 Subject: [PATCH 117/138] Organize report rules and paths Harmonize use of output/report and workflow/scripts/report directories for report-specific stuff. --- workflow/rules/distances.smk | 6 +- workflow/rules/evolution.smk | 2 +- workflow/rules/report.smk | 55 +++++++------------ workflow/rules/vaf.smk | 13 +++++ ...ap.R => pairwise_trajectory_correlation.R} | 0 .../{window.py => report/window_data.py} | 0 6 files changed, 38 insertions(+), 38 deletions(-) rename workflow/scripts/{report/heatmap.R => pairwise_trajectory_correlation.R} (100%) rename workflow/scripts/{window.py => report/window_data.py} (100%) diff --git a/workflow/rules/distances.smk b/workflow/rules/distances.smk index 833589a..abbf97b 100644 --- a/workflow/rules/distances.smk +++ b/workflow/rules/distances.smk @@ -27,7 +27,7 @@ rule afwdist_weighted_distances: variants = OUTDIR/f"{OUTPUT_NAME}.variants.afwdist.csv", reference = OUTDIR/f"{OUTPUT_NAME}.ancestor.fasta", output: - distances = temp(REPORT_DIR_TABLES/"distances.raw.csv"), + distances = temp(OUTDIR/"distances.raw.csv"), log: LOGDIR/"afwdist_weighted_distances"/"log.txt" shell: @@ -43,9 +43,9 @@ rule format_afwdist_results: params: samples = sorted(iter_samples()), input: - distances = REPORT_DIR_TABLES/"distances.raw.csv", + distances = OUTDIR/"distances.raw.csv", output: - distances = REPORT_DIR_TABLES/"distances.csv", + distances = OUTDIR/"distances.csv", log: LOGDIR/"format_afwdist_results"/"log.txt" script: diff --git a/workflow/rules/evolution.smk b/workflow/rules/evolution.smk index ee7156d..5aeaa8d 100644 --- a/workflow/rules/evolution.smk +++ b/workflow/rules/evolution.smk @@ -38,7 +38,7 @@ rule dnds_data: variants = OUTDIR/f"{OUTPUT_NAME}.variants.tsv", metadata = config["METADATA"] output: - table = report(REPORT_DIR_TABLES/"dnds.csv") + table = report(OUTDIR/"dnds.csv") log: LOGDIR / "dnds_data" / "log.txt" script: diff --git a/workflow/rules/report.smk b/workflow/rules/report.smk index c8ee402..a4f46cc 100644 --- a/workflow/rules/report.smk +++ b/workflow/rules/report.smk @@ -27,38 +27,6 @@ rule demix_plot: "../scripts/report/demix_plot.R" -rule heatmap_plot_data: - conda: "../envs/renv.yaml" - input: - variants = OUTDIR/f"{OUTPUT_NAME}.variants.tsv", - metadata = config["METADATA"] - output: - table = report(REPORT_DIR_TABLES/"heatmap.csv") - log: - LOGDIR / "heatmap" / "log.txt" - script: - "../scripts/report/heatmap.R" - - -rule window: - conda: "../envs/biopython.yaml" - params: - window = config["WINDOW"]["WIDTH"], - step = config["WINDOW"]["STEP"], - features = config.get("GB_FEATURES", {}), - gb_qualifier_display = "gene" - input: - variants = OUTDIR/f"{OUTPUT_NAME}.variants.tsv", - gb = OUTDIR/"reference.gb", - output: - window_df = temp(REPORT_DIR_TABLES/"window.csv"), - json = temp(REPORT_DIR_TABLES/"window.json"), - log: - LOGDIR / "window" / "log.txt" - script: - "../scripts/window.py" - - rule diversity_data: threads: 4 conda: "../envs/renv.yaml" @@ -142,6 +110,25 @@ rule polymorphic_sites_over_time_plot: "../scripts/report/polymorphic_sites_over_time_plot.R" +rule window_data: + conda: "../envs/biopython.yaml" + params: + window = config["WINDOW"]["WIDTH"], + step = config["WINDOW"]["STEP"], + features = config.get("GB_FEATURES", {}), + gb_qualifier_display = "gene" + input: + variants = OUTDIR/f"{OUTPUT_NAME}.variants.tsv", + gb = OUTDIR/"reference.gb", + output: + window_df = temp(REPORT_DIR_TABLES/"window.csv"), + json = temp(REPORT_DIR_TABLES/"window.json"), + log: + LOGDIR / "window_data" / "log.txt" + script: + "../scripts/report/window_data.py" + + rule nv_panel_data: conda: "../envs/renv.yaml" input: @@ -275,7 +262,7 @@ rule allele_freq_tree_data: use_bionj = config["USE_BIONJ"], ref_name = config["ALIGNMENT_REFERENCE"], input: - dist = REPORT_DIR_TABLES/"distances.csv", + dist = OUTDIR/"distances.csv", output: tree = report(REPORT_DIR_TABLES/"allele_freq_tree.nwk"), log: @@ -434,9 +421,9 @@ rule report: panel = report(REPORT_DIR_PLOTS/"af_trajectory_panel.png"), tree = report(REPORT_DIR_PLOTS/"allele_freq_tree.png"), temest = report(REPORT_DIR_PLOTS/"time_signal.png"), - heat_table = report(REPORT_DIR_TABLES/"heatmap.csv"), evo = report(REPORT_DIR_PLOTS/"dn_and_ds.png"), omega_plot = report(REPORT_DIR_PLOTS/"dnds.png"), + heat_table = report(OUTDIR/"vaf"/"pairwise_trajectory_correlation.csv"), freyja_ts = OUTDIR/"demixing"/"freyja_data"/"last_barcode_update.txt", value = REPORT_DIR_TABLES/"diversity.json", stats_lm = REPORT_DIR_TABLES/"time_signal.json", diff --git a/workflow/rules/vaf.smk b/workflow/rules/vaf.smk index ea5c7b9..09238e4 100644 --- a/workflow/rules/vaf.smk +++ b/workflow/rules/vaf.smk @@ -203,3 +203,16 @@ use rule concat_vcf_fields as concat_variants with: expand(OUTDIR/"vaf"/"{sample}.variants.tsv", sample=iter_samples()), output: OUTDIR/f"{OUTPUT_NAME}.variants.tsv", + + +rule pairwise_trajectory_correlation: + conda: "../envs/renv.yaml" + input: + variants = OUTDIR/f"{OUTPUT_NAME}.variants.tsv", + metadata = config["METADATA"], + output: + table = report(OUTDIR/"vaf"/"pairwise_trajectory_correlation.csv"), + log: + LOGDIR / "pairwise_trajectory_correlation" / "log.txt" + script: + "../scripts/report/pairwise_trajectory_correlation.R" diff --git a/workflow/scripts/report/heatmap.R b/workflow/scripts/pairwise_trajectory_correlation.R similarity index 100% rename from workflow/scripts/report/heatmap.R rename to workflow/scripts/pairwise_trajectory_correlation.R diff --git a/workflow/scripts/window.py b/workflow/scripts/report/window_data.py similarity index 100% rename from workflow/scripts/window.py rename to workflow/scripts/report/window_data.py From 9def7f5f0362c5418a4b923f2277a740a1cf6d62 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 4 Nov 2025 18:12:34 +0100 Subject: [PATCH 118/138] Update report and temp usage --- workflow/rules/report.smk | 14 +++++++------- 1 file changed, 7 insertions(+), 7 deletions(-) diff --git a/workflow/rules/report.smk b/workflow/rules/report.smk index a4f46cc..328d0c1 100644 --- a/workflow/rules/report.smk +++ b/workflow/rules/report.smk @@ -4,7 +4,7 @@ rule demix_plot_data: summary_demixing = OUTDIR/"demixing"/"summary.csv", metadata = config["METADATA"] output: - data = report(REPORT_DIR_TABLES/"demix.csv") + data = REPORT_DIR_TABLES/"demix.csv" log: LOGDIR / "demix_plot_data" / "log.txt" script: @@ -121,7 +121,7 @@ rule window_data: variants = OUTDIR/f"{OUTPUT_NAME}.variants.tsv", gb = OUTDIR/"reference.gb", output: - window_df = temp(REPORT_DIR_TABLES/"window.csv"), + window_df = REPORT_DIR_TABLES/"window.csv", json = temp(REPORT_DIR_TABLES/"window.json"), log: LOGDIR / "window_data" / "log.txt" @@ -264,7 +264,7 @@ rule allele_freq_tree_data: input: dist = OUTDIR/"distances.csv", output: - tree = report(REPORT_DIR_TABLES/"allele_freq_tree.nwk"), + tree = REPORT_DIR_TABLES/"allele_freq_tree.nwk", log: LOGDIR / "allele_freq_tree_data" / "log.txt" script: @@ -299,7 +299,7 @@ rule time_signal_data: metadata = config["METADATA"], output: table = report(REPORT_DIR_TABLES/"time_signal.csv"), - json = REPORT_DIR_TABLES/"time_signal.json" + json = REPORT_DIR_TABLES/"time_signal.json", log: LOGDIR / "time_signal_data" / "log.txt" script: @@ -348,7 +348,7 @@ rule af_time_correlation_data: variants = OUTDIR/f"{OUTPUT_NAME}.variants.tsv", metadata = config["METADATA"], output: - fmt_variants = temp(OUTDIR/f"{OUTPUT_NAME}.variants.filled.dated.tsv"), + fmt_variants = temp(REPORT_DIR_TABLES/"variants.filled.dated.tsv"), correlations = report(REPORT_DIR_TABLES/"af_time_correlation.csv"), subset = REPORT_DIR_TABLES/"af_time_correlation.subset.txt", log: @@ -382,7 +382,7 @@ rule af_trajectory_panel_plot: plot_width_mm = 159.2, random_color_seed = 7291, input: - fmt_variants = OUTDIR/f"{OUTPUT_NAME}.variants.filled.dated.tsv", + fmt_variants = REPORT_DIR_TABLES/"variants.filled.dated.tsv", subset = REPORT_DIR_TABLES/"af_time_correlation.subset.txt" output: plot = report(REPORT_DIR_PLOTS/"af_trajectory_panel.png"), @@ -398,7 +398,7 @@ rule summary_table: report = report(OUTDIR/f"{OUTPUT_NAME}.lineage_report.csv"), metadata = config["METADATA"] output: - table = temp(OUTDIR/"summary_table.csv") + table = REPORT_DIR_TABLES/"summary_table.csv" log: LOGDIR / "summary_table" / "log.txt" script: From 79734905eb6016e109cf5b46597841ce435fdb89 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 4 Nov 2025 18:40:06 +0100 Subject: [PATCH 119/138] Rename variant calling config options Also adds demix option to restrict the maximum depth passed to samtools mpileup --- .test/targets.yaml | 6 ++---- config/README.md | 10 +++++----- config/config.yaml | 10 +++++----- workflow/rules/demix.smk | 3 ++- workflow/rules/report.smk | 6 +++--- workflow/rules/vaf.smk | 16 ++++++++-------- 6 files changed, 25 insertions(+), 26 deletions(-) diff --git a/.test/targets.yaml b/.test/targets.yaml index dfbcb7c..bac704f 100644 --- a/.test/targets.yaml +++ b/.test/targets.yaml @@ -23,11 +23,9 @@ PLOTS: PROBLEMATIC_VCF: "https://raw.githubusercontent.com/W-L/ProblematicSites_SARS-CoV2/da322c32004f7b16bdaa6a8ee7fd24d56e79d9dc/problematic_sites_sarsCov2.vcf" VC: - MAX_DEPTH: 0 MIN_QUALITY: 0 - IVAR_QUALITY: 0 - IVAR_FREQ: 0.05 - IVAR_DEPTH: 5 + MIN_FREQ: 0.05 + MIN_DEPTH: 5 DEMIX: MIN_QUALITY: 0 COV_CUTOFF: 0 diff --git a/config/README.md b/config/README.md index 14fa5aa..8ca4afd 100644 --- a/config/README.md +++ b/config/README.md @@ -140,14 +140,14 @@ All of the following variables are pre-defined in [config.yaml](/config/config.y - `REPS`: number of replicates. - `THRESHOLD`: value cutoff for visualization. - `VC`: variant calling configuration: - - `MAX_DEPTH`: maximum depth at a position for `samtools mpileup` (option `-d`). - - `MIN_QUALITY`: minimum base quality for `samtools mpileup` (option `-Q`). - - `IVAR_QUALITY`: minimum base quality for `ivar variants` (option `-q`). - - `IVAR_FREQ`: minimum frequency threshold for `ivar variants` (option `-t`). - - `IVAR_DEPTH`: minimum read depth for `ivar variants` (option `-m`). + - `MIN_QUALITY`: minimum base quality for `ivar variants` (option `-q`). + - `MIN_FREQ`: minimum frequency threshold for `ivar variants` (option `-t`). + - `MIN_DEPTH`: minimum read depth for `ivar variants` (option `-m`). + - `MAX_DEPTH`: maximum read depth for `samtools mpileup` (option `-d`). - `DEMIX`: demixing configuration (uses [Freyja](https://github.com/andersen-lab/Freyja), see also [its docs](https://andersen-lab.github.io/Freyja/index.html)): - `PATHOGEN`: pathogen of interest for `freyja update` (option `--pathogen`); must be 'SARS-CoV-2'. - `MIN_QUALITY`: minimum quality for `freyja variants` (option `--minq`). + - `MAX_DEPTH`: maximum read depth for `samtools mpileup` (option `-d`). - `COV_CUTOFF`: minimum depth to calculate the reported "coverage" (percent of sites with that depth) for `freyja demix` (option `--covcut`). - `MIN_ABUNDANCE`: minimum lineage estimated abundance for `freyja demix` (option `--eps`). - `CONFIRMED_ONLY`: exclude unconfirmed lineages in `freyja demix` (option `--confirmedonly`). diff --git a/config/config.yaml b/config/config.yaml index c9e0373..f8b7a98 100644 --- a/config/config.yaml +++ b/config/config.yaml @@ -13,11 +13,10 @@ SHALRT: REPS: 1000 THRESHOLD: 0.80 VC: - MAX_DEPTH: 0 - MIN_QUALITY: 0 - IVAR_QUALITY: 20 - IVAR_FREQ: 0.05 - IVAR_DEPTH: 30 + MIN_QUALITY: 20 + MIN_FREQ: 0.05 + MIN_DEPTH: 30 + MAX_DEPTH: 1000000 ANNOTATION: # see: https://pcingola.github.io/SnpEff/adds/VCFannotationformat_v1.0.pdf SNPEFF_COLS: @@ -52,6 +51,7 @@ ANNOTATION: DEMIX: PATHOGEN: "SARS-CoV-2" MIN_QUALITY: 20 + MAX_DEPTH: 1000000 COV_CUTOFF: 30 MIN_ABUNDANCE: 0.0001 CONFIRMED_ONLY: false diff --git a/workflow/rules/demix.smk b/workflow/rules/demix.smk index 367e7b1..4973b3e 100644 --- a/workflow/rules/demix.smk +++ b/workflow/rules/demix.smk @@ -28,6 +28,7 @@ rule demix_preprocessing: bam = get_input_bam, ref_fasta = lambda wildcards: select_mapping_references_fasta() params: + max_depth = config["DEMIX"]["MAX_DEPTH"], minq = config["DEMIX"]["MIN_QUALITY"], output: depth_file = OUTDIR/"demixing"/"{sample}/{sample}_depth.txt", @@ -37,7 +38,7 @@ rule demix_preprocessing: ivar = LOGDIR / "demix_preprocessing" / "{sample}_ivar.log.txt", shell: "set -euo pipefail && " - "samtools mpileup -aa -A -d 600000 -Q {params.minq} -q 0 -B -f {input.ref_fasta:q} {input.bam:q} >sample.pileup 2>{log.pileup:q} && " + "samtools mpileup -aa -A -d {params.max_depth} -Q {params.minq} -q 0 -B -f {input.ref_fasta:q} {input.bam:q} >sample.pileup 2>{log.pileup:q} && " "ivar variants -p variants -q {params.minq} -r {input.ref_fasta:q} >{log.ivar:q} 2>&1 {output.depth_file:q} && " "mv variants.tsv {output.variants_file:q}" diff --git a/workflow/rules/report.smk b/workflow/rules/report.smk index 328d0c1..64b3ecf 100644 --- a/workflow/rules/report.smk +++ b/workflow/rules/report.smk @@ -81,7 +81,7 @@ rule extract_genbank_regions: rule polymorphic_sites_over_time_data: conda: "../envs/renv.yaml" params: - max_alt_freq = 1.0 - config["VC"]["IVAR_FREQ"], + max_alt_freq = 1.0 - config["VC"]["MIN_FREQ"], input: variants = OUTDIR/f"{OUTPUT_NAME}.variants.tsv", metadata = config["METADATA"], @@ -428,11 +428,11 @@ rule report: value = REPORT_DIR_TABLES/"diversity.json", stats_lm = REPORT_DIR_TABLES/"time_signal.json", stats_ml = REPORT_DIR_TABLES/"context_phylogeny.json", - table = OUTDIR/"summary_table.csv", + table = REPORT_DIR_TABLES/"summary_table.csv", sum_nv = REPORT_DIR_TABLES/"nv_panel_summary.json", params: workflow_version = get_repo_version(BASE_PATH.as_posix(), __version__), - min_ivar_freq = config["VC"]["IVAR_FREQ"], + min_ivar_freq = config["VC"]["MIN_FREQ"], ufboot_reps = config["UFBOOT"]["REPS"], shalrt_reps = config["SHALRT"]["REPS"], name = config["OUTPUT_NAME"], diff --git a/workflow/rules/vaf.smk b/workflow/rules/vaf.smk index 09238e4..284265b 100644 --- a/workflow/rules/vaf.smk +++ b/workflow/rules/vaf.smk @@ -4,11 +4,11 @@ rule snps_to_ancestor: shadow: "minimal" conda: "../envs/var_calling.yaml" params: - max_depth = config["VC"]["MAX_DEPTH"], - min_quality = config["VC"]["MIN_QUALITY"], - ivar_quality = config["VC"]["IVAR_QUALITY"], - ivar_freq = config["VC"]["IVAR_FREQ"], - ivar_depth = config["VC"]["IVAR_DEPTH"] + mpileup_depth = config["VC"]["MAX_DEPTH"], + mpileup_quality = 0, + ivar_quality = config["VC"]["MIN_QUALITY"], + ivar_freq = config["VC"]["MIN_FREQ"], + ivar_depth = config["VC"]["MIN_DEPTH"], input: reference_fasta = OUTDIR/f"{OUTPUT_NAME}.ancestor.fasta", bam = get_input_bam, @@ -35,10 +35,10 @@ rule snps_to_ancestor: samtools mpileup \ -aa \ --ignore-overlaps \ - -d {params.max_depth} \ + -d {params.mpileup_depth} \ --count-orphans \ --no-BAQ \ - -Q {params.min_quality} \ + -Q {params.mpileup_quality} \ -f renamed_reference.fasta \ {input.bam} \ | ivar variants \ @@ -215,4 +215,4 @@ rule pairwise_trajectory_correlation: log: LOGDIR / "pairwise_trajectory_correlation" / "log.txt" script: - "../scripts/report/pairwise_trajectory_correlation.R" + "../scripts/pairwise_trajectory_correlation.R" From 72d4959567f1a3e731a7445e7ef5addc67a5ad21 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 4 Nov 2025 18:43:43 +0100 Subject: [PATCH 120/138] Update Dockerfile --- Dockerfile | 86 ++++++++++++++++++++++++++++-------------------------- 1 file changed, 45 insertions(+), 41 deletions(-) diff --git a/Dockerfile b/Dockerfile index 7f39843..ce29cbd 100644 --- a/Dockerfile +++ b/Dockerfile @@ -1,12 +1,23 @@ FROM condaforge/miniforge3:latest LABEL io.github.snakemake.containerized="true" -LABEL io.github.snakemake.conda_env_hash="bd9b69b913e7b4e17d7d5ca7169a5815c3145ea775af87163f18c0b92abc1bf8" +LABEL io.github.snakemake.conda_env_hash="d883c5236d6779c015257dd3adab712fee0805655dfc5ccdc99b24758b7249a5" # Step 2: Retrieve conda environments +# Conda environment: +# source: workflow/envs/afwdist.yaml +# prefix: /conda-envs/9c24a867826615972cc288081976e7fc +# channels: +# - bioconda +# - conda-forge +# dependencies: +# - afwdist==1.0.0 +RUN mkdir -p /conda-envs/9c24a867826615972cc288081976e7fc +COPY workflow/envs/afwdist.yaml /conda-envs/9c24a867826615972cc288081976e7fc/environment.yaml + # Conda environment: # source: workflow/envs/biopython.yaml -# prefix: /conda-envs/bd81c49fcb540d7706807c1683ba7200 +# prefix: /conda-envs/162796cecea22d99c8702138f0c48e2f # channels: # - conda-forge # - bioconda @@ -14,12 +25,8 @@ LABEL io.github.snakemake.conda_env_hash="bd9b69b913e7b4e17d7d5ca7169a5815c3145e # - python==3.10 # - biopython==1.81 # - pandas==2.0.3 -# - pip==23.2.1 -# - mafft==7.525 -# - pip: -# - gb2seq==0.2.20 -RUN mkdir -p /conda-envs/bd81c49fcb540d7706807c1683ba7200 -COPY workflow/envs/biopython.yaml /conda-envs/bd81c49fcb540d7706807c1683ba7200/environment.yaml +RUN mkdir -p /conda-envs/162796cecea22d99c8702138f0c48e2f +COPY workflow/envs/biopython.yaml /conda-envs/162796cecea22d99c8702138f0c48e2f/environment.yaml # Conda environment: # source: workflow/envs/fetch.yaml @@ -35,14 +42,14 @@ COPY workflow/envs/fetch.yaml /conda-envs/9439457f932a4fbca3665c9ea1ac2f0a/envir # Conda environment: # source: workflow/envs/freyja.yaml -# prefix: /conda-envs/ee7a2e1b4ec9a7a9999f34dddaea0605 +# prefix: /conda-envs/bb4c5f3a509433cc08861582fab4a705 # channels: # - conda-forge # - bioconda # dependencies: -# - freyja==1.4.2 -RUN mkdir -p /conda-envs/ee7a2e1b4ec9a7a9999f34dddaea0605 -COPY workflow/envs/freyja.yaml /conda-envs/ee7a2e1b4ec9a7a9999f34dddaea0605/environment.yaml +# - freyja==2.0.1 +RUN mkdir -p /conda-envs/bb4c5f3a509433cc08861582fab4a705 +COPY workflow/envs/freyja.yaml /conda-envs/bb4c5f3a509433cc08861582fab4a705/environment.yaml # Conda environment: # source: workflow/envs/gisaidr.yaml @@ -56,7 +63,6 @@ COPY workflow/envs/freyja.yaml /conda-envs/ee7a2e1b4ec9a7a9999f34dddaea0605/envi # - r-logger==0.2.2 RUN mkdir -p /conda-envs/3fad3c9cdfa40bee9404f6a2e8fda69f COPY workflow/envs/gisaidr.yaml /conda-envs/3fad3c9cdfa40bee9404f6a2e8fda69f/environment.yaml -COPY workflow/envs/gisaidr.post-deploy.sh /conda-envs/3fad3c9cdfa40bee9404f6a2e8fda69f/post-deploy.sh # Conda environment: # source: workflow/envs/iqtree.yaml @@ -93,56 +99,53 @@ COPY workflow/envs/pangolin.yaml /conda-envs/fd645c541ee7a3d43fb9167441b77888/en # Conda environment: # source: workflow/envs/quarto_render.yaml -# prefix: /conda-envs/f2a098519cf1f8c4cecb3c13f8c92883 +# prefix: /conda-envs/b33b29b10147e6767116c7a41920d484 # channels: # - conda-forge -# - bioconda # dependencies: -# - r-base==4.3.1 -# - r-gt==0.9.0 -# - quarto==1.3.450 -# - r-jsonlite==1.8.5 +# - r-base==4.5.2 +# - r-gt==1.1.0 +# - quarto==1.8.25 +# - deno==2.3.1 # - r-tidyverse==2.0.0 -# - r-quarto==1.2 -# - r-heatmaply==1.4.2 -# - r-readr==2.1.4 -RUN mkdir -p /conda-envs/f2a098519cf1f8c4cecb3c13f8c92883 -COPY workflow/envs/quarto_render.yaml /conda-envs/f2a098519cf1f8c4cecb3c13f8c92883/environment.yaml +# - r-quarto==1.5.1 +# - r-heatmaply==1.6.0 +RUN mkdir -p /conda-envs/b33b29b10147e6767116c7a41920d484 +COPY workflow/envs/quarto_render.yaml /conda-envs/b33b29b10147e6767116c7a41920d484/environment.yaml # Conda environment: # source: workflow/envs/renv.yaml -# prefix: /conda-envs/4b57bfc237ddc217c1f0b04d34dc06ef +# prefix: /conda-envs/8ad6cdcf265d30289788da99d5bf9fff # channels: # - conda-forge # - bioconda # dependencies: -# - r-base=4.1.3 +# - r-base=4.3.2 # - r-tidyverse==2.0.0 # - r-ggrepel==0.9.3 -# - r-stringi==1.7.12 # - r-ggpubr==0.6.0 -# - bioconductor-ggtree==3.2.0 -# - r-ape==5.7 +# - bioconductor-ggtree==3.10.0 +# - r-ape==5.8 # - r-adephylo==1.1_13 # - r-pegas==1.2 # - r-data.table==1.14.8 # - r-future.apply==1.11.0 -# - r-scales==1.2.1 +# - r-scales==1.3.0 # - r-showtext==0.9_6 -# - r-jsonlite==1.8.5 # - r-logger==0.2.2 -RUN mkdir -p /conda-envs/4b57bfc237ddc217c1f0b04d34dc06ef -COPY workflow/envs/renv.yaml /conda-envs/4b57bfc237ddc217c1f0b04d34dc06ef/environment.yaml +RUN mkdir -p /conda-envs/8ad6cdcf265d30289788da99d5bf9fff +COPY workflow/envs/renv.yaml /conda-envs/8ad6cdcf265d30289788da99d5bf9fff/environment.yaml # Conda environment: # source: workflow/envs/snpeff.yaml -# prefix: /conda-envs/1934df0e4df02a7ee33c52f53f9e3c30 +# prefix: /conda-envs/0adafb79cb1bec58ef4c77bf4cca4f95 # channels: # - bioconda # dependencies: # - snpeff==5.1d -RUN mkdir -p /conda-envs/1934df0e4df02a7ee33c52f53f9e3c30 -COPY workflow/envs/snpeff.yaml /conda-envs/1934df0e4df02a7ee33c52f53f9e3c30/environment.yaml +# - snpsift==5.1d +RUN mkdir -p /conda-envs/0adafb79cb1bec58ef4c77bf4cca4f95 +COPY workflow/envs/snpeff.yaml /conda-envs/0adafb79cb1bec58ef4c77bf4cca4f95/environment.yaml # Conda environment: # source: workflow/envs/var_calling.yaml @@ -158,16 +161,17 @@ COPY workflow/envs/var_calling.yaml /conda-envs/5150d0f0a91d7f7a789a06f453d63479 # Step 3: Generate conda environments -RUN conda env create --prefix /conda-envs/bd81c49fcb540d7706807c1683ba7200 --file /conda-envs/bd81c49fcb540d7706807c1683ba7200/environment.yaml && \ +RUN conda env create --prefix /conda-envs/9c24a867826615972cc288081976e7fc --file /conda-envs/9c24a867826615972cc288081976e7fc/environment.yaml && \ + conda env create --prefix /conda-envs/162796cecea22d99c8702138f0c48e2f --file /conda-envs/162796cecea22d99c8702138f0c48e2f/environment.yaml && \ conda env create --prefix /conda-envs/9439457f932a4fbca3665c9ea1ac2f0a --file /conda-envs/9439457f932a4fbca3665c9ea1ac2f0a/environment.yaml && \ - conda env create --prefix /conda-envs/ee7a2e1b4ec9a7a9999f34dddaea0605 --file /conda-envs/ee7a2e1b4ec9a7a9999f34dddaea0605/environment.yaml && \ + conda env create --prefix /conda-envs/bb4c5f3a509433cc08861582fab4a705 --file /conda-envs/bb4c5f3a509433cc08861582fab4a705/environment.yaml && \ conda env create --prefix /conda-envs/3fad3c9cdfa40bee9404f6a2e8fda69f --file /conda-envs/3fad3c9cdfa40bee9404f6a2e8fda69f/environment.yaml && \ conda env create --prefix /conda-envs/0a608afb24723cb6fa8aef748f5efbc8 --file /conda-envs/0a608afb24723cb6fa8aef748f5efbc8/environment.yaml && \ conda env create --prefix /conda-envs/04a3347f94ddf7e21c34bc49e5246076 --file /conda-envs/04a3347f94ddf7e21c34bc49e5246076/environment.yaml && \ conda env create --prefix /conda-envs/fd645c541ee7a3d43fb9167441b77888 --file /conda-envs/fd645c541ee7a3d43fb9167441b77888/environment.yaml && \ - conda env create --prefix /conda-envs/f2a098519cf1f8c4cecb3c13f8c92883 --file /conda-envs/f2a098519cf1f8c4cecb3c13f8c92883/environment.yaml && \ - conda env create --prefix /conda-envs/4b57bfc237ddc217c1f0b04d34dc06ef --file /conda-envs/4b57bfc237ddc217c1f0b04d34dc06ef/environment.yaml && \ - conda env create --prefix /conda-envs/1934df0e4df02a7ee33c52f53f9e3c30 --file /conda-envs/1934df0e4df02a7ee33c52f53f9e3c30/environment.yaml && \ + conda env create --prefix /conda-envs/b33b29b10147e6767116c7a41920d484 --file /conda-envs/b33b29b10147e6767116c7a41920d484/environment.yaml && \ + conda env create --prefix /conda-envs/8ad6cdcf265d30289788da99d5bf9fff --file /conda-envs/8ad6cdcf265d30289788da99d5bf9fff/environment.yaml && \ + conda env create --prefix /conda-envs/0adafb79cb1bec58ef4c77bf4cca4f95 --file /conda-envs/0adafb79cb1bec58ef4c77bf4cca4f95/environment.yaml && \ conda env create --prefix /conda-envs/5150d0f0a91d7f7a789a06f453d63479 --file /conda-envs/5150d0f0a91d7f7a789a06f453d63479/environment.yaml && \ conda clean --all -y From f0622c0e31ea0eaf1118218af6220cf0b3e4668b Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 4 Nov 2025 18:57:56 +0100 Subject: [PATCH 121/138] Add tl;dr to instructions --- config/README.md | 10 ++++++++++ 1 file changed, 10 insertions(+) diff --git a/config/README.md b/config/README.md index 8ca4afd..9ca7c5c 100644 --- a/config/README.md +++ b/config/README.md @@ -12,6 +12,16 @@ For example, existing [profiles](https://snakemake.readthedocs.io/en/stable/gett are cross-compatible as well, but note that the `--use-conda` flag is deprecated starting with Snakemake 8. Instead, use `--software-deployment-method conda`. +## tl;dr + +- **Needs**: + - Snakemake 7.19 or later. + - One FASTA per sample. + - One BAM per sample. + - One metadata CSV with columns `ID`, `CollectionDate` (YYYY-MM-DD), `ResidenceCity` and `GISAIDEPI` (can be empty). +- **Setup**: edit [targets.yaml](/config/targets.yaml) (set `SAMPLES` and `METADATA`, at least) or build it using [`build_targets.py`](/build_targets.py). Leave `CONTEXT_FASTA: null` to auto-download from GISAID (needs `config/gisaid.yaml` with your username and password) or set a local FASTA path if download fails (see GISAID disclaimer). +- **Run**: `snakemake (--use-conda | --sdm conda) -c4`. + ## Inputs and outputs The workflow requires a set of FASTA files (one per target sample), a corresponding set of From c1d4c17a46fb92a96ee08de5fceb7718ab2c28e9 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Wed, 5 Nov 2025 09:52:17 +0100 Subject: [PATCH 122/138] Update dN/dS rule name and paths --- workflow/rules/evolution.smk | 8 ++++---- workflow/rules/report.smk | 2 +- workflow/scripts/{dnds_data.R => calculate_dnds.R} | 0 3 files changed, 5 insertions(+), 5 deletions(-) rename workflow/scripts/{dnds_data.R => calculate_dnds.R} (100%) diff --git a/workflow/rules/evolution.smk b/workflow/rules/evolution.smk index 5aeaa8d..0380940 100644 --- a/workflow/rules/evolution.smk +++ b/workflow/rules/evolution.smk @@ -31,15 +31,15 @@ rule n_s_sites: "../scripts/n_s_sites_from_fasta.py" -rule dnds_data: +rule calculate_dnds: conda: "../envs/renv.yaml" input: n_s_sites = OUTDIR/f"{OUTPUT_NAME}.ancestor.n_s.sites.csv", variants = OUTDIR/f"{OUTPUT_NAME}.variants.tsv", metadata = config["METADATA"] output: - table = report(OUTDIR/"dnds.csv") + table = OUTDIR/f"{OUTPUT_NAME}.dnds.csv", log: - LOGDIR / "dnds_data" / "log.txt" + LOGDIR / "calculate_dnds" / "log.txt" script: - "../scripts/dnds_data.R" + "../scripts/calculate_dnds.R" diff --git a/workflow/rules/report.smk b/workflow/rules/report.smk index 64b3ecf..cf82fb5 100644 --- a/workflow/rules/report.smk +++ b/workflow/rules/report.smk @@ -329,7 +329,7 @@ rule dnds_plots: plot_height_mm = 119.4, plot_width_mm = 159.2, input: - table = REPORT_DIR_TABLES/"dnds.csv", + table = OUTDIR/f"{OUTPUT_NAME}.dnds.csv", output: plot_dn_ds = report(REPORT_DIR_PLOTS/"dn_and_ds.png"), plot_omega = report(REPORT_DIR_PLOTS/"dnds.png"), diff --git a/workflow/scripts/dnds_data.R b/workflow/scripts/calculate_dnds.R similarity index 100% rename from workflow/scripts/dnds_data.R rename to workflow/scripts/calculate_dnds.R From 981165c25e3e915bb0a7a9f4014610ffc53634ea Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Wed, 5 Nov 2025 10:27:20 +0100 Subject: [PATCH 123/138] Add instructions of SnpEff config fields Also updates the order in the configuration file --- config/README.md | 6 +++++ config/config.yaml | 62 +++++++++++++++++++++++----------------------- 2 files changed, 37 insertions(+), 31 deletions(-) diff --git a/config/README.md b/config/README.md index 9ca7c5c..43f7e5e 100644 --- a/config/README.md +++ b/config/README.md @@ -180,6 +180,12 @@ All of the following variables are pre-defined in [config.yaml](/config/config.y After `INCLUDE` is applied, any feature that matches any key/value pair in `EXCLUDE` is omitted. For example, having `EXCLUDE: {gene: [S, N]}` removes features whose `gene` qualifier equals `S` or `N`. +- `ANNOTATION`: settings for variant annotation and functional effect prediction using [SnpEff](https://pcingola.github.io/SnpEff/), which uses `ALIGNMENT_REFERENCE` for selecting the database to annotate. + - `SNPEFF_COLS`: mapping of column names (which appear in result tables) to VCF fields (extracted after annotation with [SnpSift](https://pcingola.github.io/SnpEff/#snpsift)). Some columns are hard-coded in the code, so removing them is not advised. Additional columns can be added as needed. + - `FILTER_INCLUDE` & `FILTER_EXCLUDE`: mapping of column names (from `SNPEFF_COLS`) to lists of values used for filtering the annotated variants table. `FILTER_INCLUDE` is applied first, then `FILTER_EXCLUDE`. + - `FILTER_INCLUDE`: keeps variants that match at least one listed value. + - `FILTER_EXCLUDE`: removes variant that matches any listed value. + - `VARIANT_NAME_PATTERN`: string template used to build the variant name shown in the results table (column `VARIANT_NAME`). The template is interpreted with [glue](https://glue.tidyverse.org/) and can use any column name from `SNPEFF_COLS` and some R functions. For example, `"{GENE}:{coalesce(HGVS_P, HGVS_C)}"` creates names like `S:p.D614G` (using `HGVS_P` when available, otherwise `HGVS_C`). - `GISAID`: automatic context download configuration. - `CREDENTIALS`: path of the GISAID credentials in YAML format. - `DATE_COLUMN`: name of the column that contains sampling dates (YYYY-MM-DD) in the input target metadata. diff --git a/config/config.yaml b/config/config.yaml index f8b7a98..54d5097 100644 --- a/config/config.yaml +++ b/config/config.yaml @@ -17,37 +17,6 @@ VC: MIN_FREQ: 0.05 MIN_DEPTH: 30 MAX_DEPTH: 1000000 -ANNOTATION: - # see: https://pcingola.github.io/SnpEff/adds/VCFannotationformat_v1.0.pdf - SNPEFF_COLS: - CHROM: CHROM - POS: POS - REF: REF - ALT: ALT - EFFECT: "ANN[*].EFFECT" # hard-coded column - IMPACT: "ANN[*].IMPACT" # hard-coded column - BIOTYPE: "ANN[*].BIOTYPE" - GENE: "ANN[*].GENE" # hard-coded column - GENEID: "ANN[*].GENEID" - FEATURE: "ANN[*].FEATURE" - FEATUREID: "ANN[*].FEATUREID" - HGVS_P: "ANN[*].HGVS_P" # hard-coded column - HGVS_C: "ANN[*].HGVS_C" # hard-coded column - ERRORS: "ANN[*].ERRORS" # hard-coded column - FILTER_INCLUDE: - # IMPACT: [HIGH, MODERATE, LOW] - FILTER_EXCLUDE: - EFFECT: [upstream_gene_variant, downstream_gene_variant] - ERRORS: - - ERROR_CHROMOSOME_NOT_FOUND - - ERROR_OUT_OF_CHROMOSOME_RANGE - - WARNING_REF_DOES_NOT_MATCH_GENOME - - WARNING_SEQUENCE_NOT_AVAILABLE - - WARNING_TRANSCRIPT_INCOMPLETE - - WARNING_TRANSCRIPT_MULTIPLE_STOP_CODONS - - WARNING_TRANSCRIPT_NO_START_CODON - - WARNING_TRANSCRIPT_NO_STOP_CODON - VARIANT_NAME_PATTERN: "{GENE}:{coalesce(HGVS_P, HGVS_C)}" # dplyr's coalesce finds the first non-missing element DEMIX: PATHOGEN: "SARS-CoV-2" MIN_QUALITY: 20 @@ -78,6 +47,37 @@ GB_FEATURES: - "nucleocapsid phosphoprotein" - "ORF10 protein" # EXCLUDE: ... # all +ANNOTATION: + # see: https://pcingola.github.io/SnpEff/adds/VCFannotationformat_v1.0.pdf + SNPEFF_COLS: + CHROM: CHROM + POS: POS + REF: REF + ALT: ALT + EFFECT: "ANN[*].EFFECT" + IMPACT: "ANN[*].IMPACT" + BIOTYPE: "ANN[*].BIOTYPE" + GENE: "ANN[*].GENE" + GENEID: "ANN[*].GENEID" + FEATURE: "ANN[*].FEATURE" + FEATUREID: "ANN[*].FEATUREID" + HGVS_P: "ANN[*].HGVS_P" + HGVS_C: "ANN[*].HGVS_C" + ERRORS: "ANN[*].ERRORS" + FILTER_INCLUDE: + # IMPACT: [HIGH, MODERATE, LOW] + FILTER_EXCLUDE: + EFFECT: [upstream_gene_variant, downstream_gene_variant] + ERRORS: + - ERROR_CHROMOSOME_NOT_FOUND + - ERROR_OUT_OF_CHROMOSOME_RANGE + - WARNING_REF_DOES_NOT_MATCH_GENOME + - WARNING_SEQUENCE_NOT_AVAILABLE + - WARNING_TRANSCRIPT_INCOMPLETE + - WARNING_TRANSCRIPT_MULTIPLE_STOP_CODONS + - WARNING_TRANSCRIPT_NO_START_CODON + - WARNING_TRANSCRIPT_NO_STOP_CODON + VARIANT_NAME_PATTERN: "{GENE}:{coalesce(HGVS_P, HGVS_C)}" GISAID: CREDENTIALS: "config/gisaid.yaml" DATE_COLUMN: "CollectionDate" From 3c0168a4e7d4b42447569d91554dd7402a1b534c Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Wed, 5 Nov 2025 12:20:20 +0100 Subject: [PATCH 124/138] Add --notemp to tests --- .github/workflows/test_apptainer.yml | 2 +- .github/workflows/test_v7.yml | 2 +- .github/workflows/test_v8.yml | 2 +- .github/workflows/test_v9.yml | 2 +- 4 files changed, 4 insertions(+), 4 deletions(-) diff --git a/.github/workflows/test_apptainer.yml b/.github/workflows/test_apptainer.yml index 08b8f26..452af0d 100644 --- a/.github/workflows/test_apptainer.yml +++ b/.github/workflows/test_apptainer.yml @@ -26,7 +26,7 @@ jobs: environment-file: .test/environment_apptainer.yaml - name: Run test pipeline shell: bash -el {0} - run: snakemake --snakefile .test/Snakefile --configfile config/config.yaml .test/targets.yaml -c1 --sdm apptainer conda --keep-going + run: snakemake --snakefile .test/Snakefile --configfile config/config.yaml .test/targets.yaml -c1 --sdm apptainer conda --keep-going --notemp - name: Pack logs if: success() || failure() shell: bash -el {0} diff --git a/.github/workflows/test_v7.yml b/.github/workflows/test_v7.yml index fba8bca..3416131 100644 --- a/.github/workflows/test_v7.yml +++ b/.github/workflows/test_v7.yml @@ -26,7 +26,7 @@ jobs: environment-file: .test/environment_v7.yaml - name: Run test pipeline shell: bash -el {0} - run: snakemake --snakefile .test/Snakefile --configfile config/config.yaml .test/targets.yaml --use-conda -c1 --conda-frontend conda --keep-going + run: snakemake --snakefile .test/Snakefile --configfile config/config.yaml .test/targets.yaml --use-conda -c1 --conda-frontend conda --keep-going --notemp - name: Pack logs if: success() || failure() shell: bash -el {0} diff --git a/.github/workflows/test_v8.yml b/.github/workflows/test_v8.yml index 4f4af1d..8fd912d 100644 --- a/.github/workflows/test_v8.yml +++ b/.github/workflows/test_v8.yml @@ -26,7 +26,7 @@ jobs: environment-file: .test/environment_v8.yaml - name: Run test pipeline shell: bash -el {0} - run: snakemake --snakefile .test/Snakefile --configfile config/config.yaml .test/targets.yaml --use-conda -c1 --conda-frontend conda --keep-going + run: snakemake --snakefile .test/Snakefile --configfile config/config.yaml .test/targets.yaml --use-conda -c1 --conda-frontend conda --keep-going --notemp - name: Pack logs if: success() || failure() shell: bash -el {0} diff --git a/.github/workflows/test_v9.yml b/.github/workflows/test_v9.yml index 1845334..a3e211c 100644 --- a/.github/workflows/test_v9.yml +++ b/.github/workflows/test_v9.yml @@ -26,7 +26,7 @@ jobs: environment-file: .test/environment_v9.yaml - name: Run test pipeline shell: bash -el {0} - run: snakemake --snakefile .test/Snakefile --configfile config/config.yaml .test/targets.yaml -c1 --sdm conda --keep-going + run: snakemake --snakefile .test/Snakefile --configfile config/config.yaml .test/targets.yaml -c1 --sdm conda --keep-going --notemp - name: Pack logs if: success() || failure() shell: bash -el {0} From b466f62e38ef05aedcb900dd60a12f9752149b26 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Wed, 5 Nov 2025 12:20:28 +0100 Subject: [PATCH 125/138] Update test snakefile version --- .test/Snakefile | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/.test/Snakefile b/.test/Snakefile index 66eaf84..d3ff278 100644 --- a/.test/Snakefile +++ b/.test/Snakefile @@ -8,7 +8,7 @@ import subprocess min_version("7.19") # Workflow version -__version__ = "1.2.1" +__version__ = "1.3.0" # Rules include: "../workflow/core.smk" From a1431ee6c43ff50bd22a4433b36e055085276a7b Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Wed, 5 Nov 2025 12:20:39 +0100 Subject: [PATCH 126/138] Update distances file name --- workflow/rules/distances.smk | 6 +++--- workflow/rules/report.smk | 2 +- 2 files changed, 4 insertions(+), 4 deletions(-) diff --git a/workflow/rules/distances.smk b/workflow/rules/distances.smk index abbf97b..224e48d 100644 --- a/workflow/rules/distances.smk +++ b/workflow/rules/distances.smk @@ -27,7 +27,7 @@ rule afwdist_weighted_distances: variants = OUTDIR/f"{OUTPUT_NAME}.variants.afwdist.csv", reference = OUTDIR/f"{OUTPUT_NAME}.ancestor.fasta", output: - distances = temp(OUTDIR/"distances.raw.csv"), + distances = temp(OUTDIR/f"{OUTPUT_NAME}.distances.raw.csv"), log: LOGDIR/"afwdist_weighted_distances"/"log.txt" shell: @@ -43,9 +43,9 @@ rule format_afwdist_results: params: samples = sorted(iter_samples()), input: - distances = OUTDIR/"distances.raw.csv", + distances = OUTDIR/f"{OUTPUT_NAME}.distances.raw.csv", output: - distances = OUTDIR/"distances.csv", + distances = OUTDIR/f"{OUTPUT_NAME}.distances.csv", log: LOGDIR/"format_afwdist_results"/"log.txt" script: diff --git a/workflow/rules/report.smk b/workflow/rules/report.smk index cf82fb5..ba6761b 100644 --- a/workflow/rules/report.smk +++ b/workflow/rules/report.smk @@ -262,7 +262,7 @@ rule allele_freq_tree_data: use_bionj = config["USE_BIONJ"], ref_name = config["ALIGNMENT_REFERENCE"], input: - dist = OUTDIR/"distances.csv", + dist = OUTDIR/f"{OUTPUT_NAME}.distances.csv", output: tree = REPORT_DIR_TABLES/"allele_freq_tree.nwk", log: From 73e8dc8fbaa9b4f85c01dcc189e38b6e2389233e Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Wed, 5 Nov 2025 13:50:13 +0100 Subject: [PATCH 127/138] Update variant calling threads directive This accounts for the two processes spawned by samtools and ivar, which can lead to higher than expected CPU usage --- workflow/rules/vaf.smk | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/workflow/rules/vaf.smk b/workflow/rules/vaf.smk index 284265b..2a2560b 100644 --- a/workflow/rules/vaf.smk +++ b/workflow/rules/vaf.smk @@ -1,5 +1,5 @@ rule snps_to_ancestor: - threads: 1 + threads: 2 retries: 3 shadow: "minimal" conda: "../envs/var_calling.yaml" From 677f51f5aafafe1750ad98cd62c7e632209b6c9c Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Wed, 5 Nov 2025 15:36:07 +0100 Subject: [PATCH 128/138] Rename ancestor record --- workflow/rules/asr.smk | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/workflow/rules/asr.smk b/workflow/rules/asr.smk index 7bc1c8d..d20c6d4 100644 --- a/workflow/rules/asr.smk +++ b/workflow/rules/asr.smk @@ -28,7 +28,7 @@ rule ancestor_fasta: params: node_id = "Node1", indeterminate_char = "N", - name = OUTPUT_NAME + name = "case_ancestor", input: state_file = OUTDIR/"tree"/f"{OUTPUT_NAME}.state" output: From b79c1c7e85cf92824a00d694e5dcbfffc5d30965 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Wed, 5 Nov 2025 15:38:14 +0100 Subject: [PATCH 129/138] Fix missing reference record in afwdist results Renames outgroup params for distance tree rules --- workflow/rules/distances.smk | 4 ++-- workflow/rules/report.smk | 6 +++--- workflow/scripts/format_afwdist_results.py | 1 + .../scripts/report/allele_freq_tree_data.R | 2 +- .../scripts/report/allele_freq_tree_plot.R | 18 ++++++++++-------- workflow/scripts/report/time_signal_data.R | 7 ++++--- 6 files changed, 21 insertions(+), 17 deletions(-) diff --git a/workflow/rules/distances.smk b/workflow/rules/distances.smk index 224e48d..11fae38 100644 --- a/workflow/rules/distances.smk +++ b/workflow/rules/distances.smk @@ -22,7 +22,7 @@ rule extract_afwdist_variants: rule afwdist_weighted_distances: conda: "../envs/afwdist.yaml" params: - extra_args = "" + extra_args = "", input: variants = OUTDIR/f"{OUTPUT_NAME}.variants.afwdist.csv", reference = OUTDIR/f"{OUTPUT_NAME}.ancestor.fasta", @@ -41,7 +41,7 @@ rule afwdist_weighted_distances: rule format_afwdist_results: conda: "../envs/biopython.yaml" params: - samples = sorted(iter_samples()), + samples = sorted(iter_samples()) + [config["ALIGNMENT_REFERENCE"]], input: distances = OUTDIR/f"{OUTPUT_NAME}.distances.raw.csv", output: diff --git a/workflow/rules/report.smk b/workflow/rules/report.smk index ba6761b..5c53535 100644 --- a/workflow/rules/report.smk +++ b/workflow/rules/report.smk @@ -260,7 +260,7 @@ rule allele_freq_tree_data: conda: "../envs/renv.yaml" params: use_bionj = config["USE_BIONJ"], - ref_name = config["ALIGNMENT_REFERENCE"], + outgroup_id = config["ALIGNMENT_REFERENCE"], input: dist = OUTDIR/f"{OUTPUT_NAME}.distances.csv", output: @@ -275,7 +275,7 @@ rule allele_freq_tree_plot: conda: "../envs/renv.yaml" params: design = config["PLOTS"], - ref_name = config["ALIGNMENT_REFERENCE"], + outgroup_id = config["ALIGNMENT_REFERENCE"], plot_height_mm = 119.4, plot_width_mm = 159.2, input: @@ -293,7 +293,7 @@ rule allele_freq_tree_plot: rule time_signal_data: conda: "../envs/renv.yaml" params: - ref_name = config["ALIGNMENT_REFERENCE"], + outgroup_id = config["ALIGNMENT_REFERENCE"], input: tree = report(REPORT_DIR_TABLES/"allele_freq_tree.nwk"), metadata = config["METADATA"], diff --git a/workflow/scripts/format_afwdist_results.py b/workflow/scripts/format_afwdist_results.py index ca9b24f..2f42ca6 100644 --- a/workflow/scripts/format_afwdist_results.py +++ b/workflow/scripts/format_afwdist_results.py @@ -13,6 +13,7 @@ logging.info("Initializing formatted output") output = pd.DataFrame( + data=0.0, columns=snakemake.params.samples, index=snakemake.params.samples, dtype="float64" diff --git a/workflow/scripts/report/allele_freq_tree_data.R b/workflow/scripts/report/allele_freq_tree_data.R index 7272bfa..9b20ce1 100644 --- a/workflow/scripts/report/allele_freq_tree_data.R +++ b/workflow/scripts/report/allele_freq_tree_data.R @@ -46,7 +46,7 @@ tree <- matrix %>% as.matrix() %>% as.dist() %>% tree_method() %>% - root(snakemake@params$ref_name, resolve.root = TRUE) + root(snakemake@params$outgroup_id, resolve.root = TRUE) # Resolve possible negative edge lengths in tree log_info("Resolving negative edge lengths") diff --git a/workflow/scripts/report/allele_freq_tree_plot.R b/workflow/scripts/report/allele_freq_tree_plot.R index b25404f..2a08851 100644 --- a/workflow/scripts/report/allele_freq_tree_plot.R +++ b/workflow/scripts/report/allele_freq_tree_plot.R @@ -25,16 +25,16 @@ metadata <- read_csv(snakemake@input$metadata) # Extract names from records log_debug("Reading FASTA") -study_names <- read.dna( +study_records <- read.dna( snakemake@input$study_fasta, format = "fasta", as.matrix = FALSE, ) log_debug("Extracting names") -study_names <- study_names[ - !startsWith(names(study_names), snakemake@params$ref_name) +study_records <- study_records[ + !startsWith(names(study_records), snakemake@params$outgroup_id) ] -study_names <- names(study_names) +study_names <- names(study_records) # Obtain sample names ordered by CollectionDate log_debug("Sorting names by collection date") @@ -59,19 +59,21 @@ tree_tiplab <- data.frame( ) %>% ungroup() %>% add_row( - ID = snakemake@params$ref_name, + ID = snakemake@params$outgroup_id, order = 0, - tip_label = snakemake@params$ref_name + tip_label = snakemake@params$outgroup_id ) # Read distance tree and root log_info("Reading tree") tree <- read.tree(snakemake@input$tree) %>% - root(snakemake@params$ref_name, resolve.root = TRUE) + root(snakemake@params$outgroup_id, resolve.root = TRUE) # Plot log_info("Plotting distance tree") -max.tip.length <- max(node.depth.edgelength(tree)[1:length(tree$tip.label)]) +max.tip.length <- max( + node.depth.edgelength(tree)[seq_along(length(tree$tip.label))] +) p <- ggtree(tree) %<+% tree_tiplab + geom_tiplab(aes(label = tip_label)) + geom_treescale(1.1 * max.tip.length) + diff --git a/workflow/scripts/report/time_signal_data.R b/workflow/scripts/report/time_signal_data.R index 103e95b..d209528 100644 --- a/workflow/scripts/report/time_signal_data.R +++ b/workflow/scripts/report/time_signal_data.R @@ -18,7 +18,7 @@ log_threshold(INFO) # Read distance tree and root log_info("Reading tree") tree <- read.tree(snakemake@input$tree) %>% - root(snakemake@params$ref_name, resolve.root = TRUE) + root(snakemake@params$outgroup_id, resolve.root = TRUE) # Read metadata log_info("Reading metadata") @@ -33,14 +33,15 @@ time.signal <- distRoot( ) %>% as.data.frame() %>% rownames_to_column(var = "ID") %>% - filter(ID != snakemake@params$ref_name) %>% + filter(ID != snakemake@params$outgroup_id) %>% rename(distance = ".") %>% left_join( select( metadata, ID, CollectionDate - ) + ), + by = "ID" ) %>% mutate( date_interval = as.numeric( From 2645f3e58691d0fa08c362a2740e340662bf5c7c Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Wed, 5 Nov 2025 17:00:56 +0100 Subject: [PATCH 130/138] Run quarto from command line Also fixes CSS as input --- template.qmd | 59 +++++++++++++------------- workflow/envs/quarto_render.yaml | 1 - workflow/rules/report.smk | 73 +++++++++++++++++--------------- 3 files changed, 68 insertions(+), 65 deletions(-) diff --git a/template.qmd b/template.qmd index c6aba13..90078a4 100644 --- a/template.qmd +++ b/template.qmd @@ -4,8 +4,8 @@ subtitle: "Workflow version: `r params$workflow_version`" date: last-modified date-format: "YYYY-MM-DD" title-block-banner: true -format: - html: +format: + html: page-layout: article embed-resources: true smooth-scroll: true @@ -15,36 +15,35 @@ format: toc-location: left toc-title: Summary number-sections: true -css: "`r params$css`" +css: __CSSPLACEHOLDER__ editor: source params: - css: "" - ufboot_reps: "" - shalrt_reps: "" - min_ivar_freq: "" - workflow_version: "" - use_bionj: "" - cor_method: "" - div: "" - demix: "" - tree: "" - tempest: "" - SNV: "" - SNV_s: "" - evo: "" - div_value: "" - panel: "" - volcano: "" - tree_ml: "" - fig_cor_snp: "" - stats_lm: "" - stats_ml: "" - table: "" - sum_nv: "" - heat_tab: "" - omega_plot: "" - name: "" - freyja_ts: "" + ufboot_reps: + shalrt_reps: + min_ivar_freq: + workflow_version: + use_bionj: + cor_method: + div: + demix: + tree: + tempest: + SNV: + SNV_s: + evo: + div_value: + panel: + volcano: + tree_ml: + fig_cor_snp: + stats_lm: + stats_ml: + table: + sum_nv: + heat_tab: + omega_plot: + name: + freyja_ts: output-file: report.html --- diff --git a/workflow/envs/quarto_render.yaml b/workflow/envs/quarto_render.yaml index 3df14d2..a1ab3ba 100644 --- a/workflow/envs/quarto_render.yaml +++ b/workflow/envs/quarto_render.yaml @@ -6,5 +6,4 @@ dependencies: - quarto==1.8.25 - deno==2.3.1 - r-tidyverse==2.0.0 - - r-quarto==1.5.1 - r-heatmaply==1.6.0 diff --git a/workflow/rules/report.smk b/workflow/rules/report.smk index 5c53535..77aa6aa 100644 --- a/workflow/rules/report.smk +++ b/workflow/rules/report.smk @@ -407,7 +407,7 @@ rule summary_table: rule report: conda: "../envs/quarto_render.yaml" - shadow: "shallow" + shadow: "minimal" input: qmd = Path(config["REPORT_QMD"]).resolve(), css = Path(config["REPORT_CSS"]).resolve(), @@ -443,36 +443,41 @@ rule report: log: LOGDIR / "report" / "log.txt" shell: - "set +o pipefail; " - "Rscript -e \"quarto::quarto_render(" - "input = '{input.qmd:q}', " - "execute_params=list(" - "css='{input.css:q}', " - "ufboot_reps='{params.ufboot_reps}', " - "shalrt_reps='{params.shalrt_reps}', " - "min_ivar_freq='{params.min_ivar_freq}', " - "workflow_version='{params.workflow_version}', " - "use_bionj='{params.use_bionj}', " - "cor_method='{params.cor_method}', " - "div='{input.diversity}', " - "demix ='{input.demix}', " - "tree = '{input.tree}', " - "tempest = '{input.temest}', " - "SNV = '{input.SNV}', " - "SNV_s = '{input.SNV_spike}', " - "evo = '{input.evo}', " - "div_value = '{input.value}', " - "panel = '{input.panel}', " - "volcano = '{input.volcano}', " - "tree_ml = '{input.tree_ml}', " - "fig_cor_snp = '{input.fig_cor}', " - "stats_lm = '{input.stats_lm}', " - "stats_ml = '{input.stats_ml}', " - "table = '{input.table}', " - "sum_nv = '{input.sum_nv}', " - "heat_tab = '{input.heat_table}', " - "omega_plot = '{input.omega_plot}', " - "freyja_ts = '{input.freyja_ts}', " - "name = '{params.name}'))\" " - ">{log:q} 2>&1 && " - 'mv "$(dirname {input.qmd:q})/report.html" {output.html:q}' + """ + set +o pipefail + exec >{log} && exec 2>&1 + + printf "%s\n" \ + "ufboot_reps: '{params.ufboot_reps}'" \ + "shalrt_reps: '{params.shalrt_reps}'" \ + "min_ivar_freq: '{params.min_ivar_freq}'" \ + "workflow_version: '{params.workflow_version}'" \ + "use_bionj: '{params.use_bionj}'" \ + "cor_method: '{params.cor_method}'" \ + "div: '{input.diversity}'" \ + "demix: '{input.demix}'" \ + "tree: '{input.tree}'" \ + "tempest: '{input.temest}'" \ + "SNV: '{input.SNV}'" \ + "SNV_s: '{input.SNV_spike}'" \ + "evo: '{input.evo}'" \ + "div_value: '{input.value}'" \ + "panel: '{input.panel}'" \ + "volcano: '{input.volcano}'" \ + "tree_ml: '{input.tree_ml}'" \ + "fig_cor_snp: '{input.fig_cor}'" \ + "stats_lm: '{input.stats_lm}'" \ + "stats_ml: '{input.stats_ml}'" \ + "table: '{input.table}'" \ + "sum_nv: '{input.sum_nv}'" \ + "heat_tab: '{input.heat_table}'" \ + "omega_plot: '{input.omega_plot}'" \ + "freyja_ts: '{input.freyja_ts}'" \ + "name: '{params.name}'" \ + >params.yaml + + sed "s|__CSSPLACEHOLDER__|{input.css}|g" {input.qmd:q} >report.qmd + + quarto render report.qmd --execute-params params.yaml + mv report.html 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\ No newline at end of file diff --git a/config/README.md b/config/README.md index 43f7e5e..f5cc413 100644 --- a/config/README.md +++ b/config/README.md @@ -202,24 +202,31 @@ All of the following variables are pre-defined in [config.yaml](/config/config.y - `REPORT_QMD`: path of the report template in Quarto markdown (QMD) format. - `REPORT_CSS`: path of the report stylesheet definition in CSS format. -## Workflow graphs +## Workflow visualization -To generate a simplified rule graph, run: +Snakemake enables easy visualization of workflows and rule relationships. The `--rulegraph` option outputs a DOT file that describes dependencies between rules. The example below produces an image using Graphviz: ```shell -snakemake --rulegraph | dot -Tpng > .rulegraph.png +snakemake --forceall --rulegraph | dot -Tpng >.rulegraph.png ``` ![Snakemake rule graph](/.rulegraph.png) -To generate the directed acyclic graph (DAG) of all rules -to be executed, run: +The same graph can also be rendered with other tools such as [snakevision](https://github.com/OpenOmics/snakevision) (v0.1.0). ```shell -snakemake --forceall --dag | dot -Tpng > .dag.png +snakemake --forceall --rulegraph | snakevision -s all -o .rulegraph_sv.svg ``` -![Snakemake rule graph](/.dag.png) +![Snakemake rule graph using snakevision](/.rulegraph_sv.svg) + +The `--dag` option emits an directed acyclic graph (DAG) that corresponds to the rule instances that would be executed for the current dataset. The example below produces an image using Graphviz: + +```shell +snakemake --forceall --dag | dot -Tpng >.dag.png +``` + +![Snakemake DAG](/.dag.png) ## Run modes From abe75cda8168eea46910cf112211b2451cfaa6ad Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 11 Nov 2025 13:27:37 +0100 Subject: [PATCH 132/138] Reword correlation heatmap section --- template.qmd | 19 +++++++++++++++---- 1 file changed, 15 insertions(+), 4 deletions(-) diff --git a/template.qmd b/template.qmd index 90078a4..0d680ff 100644 --- a/template.qmd +++ b/template.qmd @@ -52,6 +52,16 @@ output-file: report.html +```{r funcs, echo = F, message = F, include = F} +display.num <- function(n) { + sci <- if (n < 1e-3 || n > 1e4) { + sub("e\\+?(-?)(\\d+)", " × 10^{\\1\\2}", + formatC(n, format = "e", digits = 3)) + } else { + formatC(n, format = "f", digits = 5) + } +} +``` ```{r read, echo = F, message = F, include = F} library(jsonlite) @@ -266,12 +276,12 @@ line represents the linear model fit.](`r params$tempest`){#fig-tempest} ### Correlation between alternative alleles -To detect possible interactions between mutations, pairwise correlation between allele -frequencies have been calculated (@fig-heatmap). The heatmap is an interactive figure that allows -zooming in on specific regions. +`r cor.method.name` correlation coefficients of allele frequencies between pairs +of variants were calculated to detect interactions between them (@fig-heatmap). +The heatmap is interactive and allows zooming in on specific regions. ```{r fig-heatmap, echo = F, message = F, warning = F, fig.align = 'center'} -#| fig-cap: "Interactive hierarchically clustered heatmap of the pairwisecorrelation coefficients between the time series of allele frequencies in the case study." +#| fig-cap: "Interactive heatmap with hierarchical clustering of the pairwise correlation coefficients between the time series of allele frequencies in the case study." heatmaply_cor( cor.mat, @@ -279,6 +289,7 @@ heatmaply_cor( fontsize_row = 7, fontsize_col = 7, column_text_angle = 45, + label_names = c("row", "column", "coefficient"), width = 600, height = 600 ) From 8bd4fdc32c09d63971c1e38106057b18aaa7cbbf Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 11 Nov 2025 15:25:39 +0100 Subject: [PATCH 133/138] Update report plots labels and colors Fixes legend in context phylogeny and updates colors in diversity plot --- config/design_plots.R | 6 +++--- workflow/scripts/report/context_phylogeny_data.R | 4 ++-- workflow/scripts/report/context_phylogeny_plot.R | 4 ++-- 3 files changed, 7 insertions(+), 7 deletions(-) diff --git a/config/design_plots.R b/config/design_plots.R index fc63584..74e87d8 100644 --- a/config/design_plots.R +++ b/config/design_plots.R @@ -42,8 +42,8 @@ GENE_PALETTE <- c( # Nucleotide diversity DIVERSITY_PALETTE <- c( density_fill = "#fcbf49", - density_color = "#eae2b7", - value_color = "#d62828", + density_color = "#ffaa00", + value_color = "#D944AA", dnorm_color = "#f77f00" ) @@ -60,7 +60,7 @@ TREE_NODE_SIZE <- c( TREE_LEGEND_NAMES <- c( tip_label = "Target samples", - boot_alrt_pass = "UFBoot ≥ %.2f%s & SH-aLRT ≥ %.2f%s" + boot_alrt_pass = "UFBoot ≥ %s%s & SH-aLRT ≥ %s%s" ) # Nucleotide variants classification colors and labels diff --git a/workflow/scripts/report/context_phylogeny_data.R b/workflow/scripts/report/context_phylogeny_data.R index eb4348a..a23587d 100644 --- a/workflow/scripts/report/context_phylogeny_data.R +++ b/workflow/scripts/report/context_phylogeny_data.R @@ -20,8 +20,8 @@ source(snakemake@params[["design"]]) # Format tree label for well supported nodes TREE_LEGEND_NAMES["boot_alrt_pass"] <- sprintf( TREE_LEGEND_NAMES["boot_alrt_pass"], - snakemake@params[["boot_th"]], "%", - snakemake@params[["alrt_th"]], "%" + 100 * snakemake@params[["boot_th"]], "%", + 100 * snakemake@params[["alrt_th"]], "%" ) log_info("Reading tree") diff --git a/workflow/scripts/report/context_phylogeny_plot.R b/workflow/scripts/report/context_phylogeny_plot.R index 2aa0781..742bb24 100644 --- a/workflow/scripts/report/context_phylogeny_plot.R +++ b/workflow/scripts/report/context_phylogeny_plot.R @@ -21,8 +21,8 @@ source(snakemake@params[["design"]]) # Format tree label for well supported nodes TREE_LEGEND_NAMES["boot_alrt_pass"] <- sprintf( TREE_LEGEND_NAMES["boot_alrt_pass"], - snakemake@params[["boot_th"]], "%", - snakemake@params[["alrt_th"]], "%" + 100 * snakemake@params[["boot_th"]], "%", + 100 * snakemake@params[["alrt_th"]], "%" ) log_info("Reading JSON data") From 70322c62b5ad9216a9d972a17fdc1694beaed445 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 11 Nov 2025 15:26:28 +0100 Subject: [PATCH 134/138] Reword nucleotide diversity section and update plot --- template.qmd | 47 +++++++++++------------- workflow/scripts/report/diversity_plot.R | 16 +++++--- 2 files changed, 31 insertions(+), 32 deletions(-) diff --git a/template.qmd b/template.qmd index 0d680ff..f8c22cc 100644 --- a/template.qmd +++ b/template.qmd @@ -52,15 +52,11 @@ output-file: report.html -```{r funcs, echo = F, message = F, include = F} -display.num <- function(n) { - sci <- if (n < 1e-3 || n > 1e4) { - sub("e\\+?(-?)(\\d+)", " × 10^{\\1\\2}", - formatC(n, format = "e", digits = 3)) - } else { - formatC(n, format = "f", digits = 5) - } +```{r setup, echo = F, message = F, include = F} +display.num <- function(n, digits) { + format(n, digits = digits, nsmall = digits) } +Sys.setlocale("LC_ALL", "English") ``` ```{r read, echo = F, message = F, include = F} @@ -81,7 +77,7 @@ stats <- append( ) correlation <- stats[["r2"]] sub_rate <- stats[["sub_rate"]] -sub_rate <- round(sub_rate, digits = 2) +sub_rate <- display.num(sub_rate, 2) p_value_lm <- stats[["pvalue"]] # NV counts @@ -183,21 +179,20 @@ samples is squared.](`r params$tree_ml`){#fig-tree_ml} ### Nucleotide diversity comparison Nucleotide diversity (π) has been calculated for $`r div_values[["boot.reps"]]`$ random -sample subsets of size $`r div_values[["sample.size"]]`$, extracted -from the context dataset. The distribution of the nuclotide diversity is assumed to -`r div_values[["norm.text"]]` be normal after performing a Shapiro-Wilk test -(p-value of $`r div_values[["normal.pvalue"]]`$). - -The nucleotide diversity of the target samples is $`r format(div_values[["diversity"]], scientific = TRUE) `$ (vertical line in @fig-div). -Assuming the independence of the context samples, the `r div_values[["type.test"]]` -p-value of the context samples having a nucleotide diversity (smooth curve in @fig-div) -as low as that of the target dataset is $`r div_values[["p.value"]]`$. - -![Analysis of the nucleotide diversity (π). The smooth curve describes -a normal distribution with the same mean and standard deviation as the distribution -of π from $`r div_values[["boot.reps"]]`$ subsets of $`r div_values[["sample.size"]]`$ -sequences from the context. The vertical line indicates the π value of the -target samples.](`r params$div`){#fig-div} +subsets of size $`r div_values[["sample.size"]]`$ drawn from the context dataset. +A Shapiro–Wilk test indicates the distribution is `r div_values[["norm.text"]]` +normal (p-value of $`r div_values[["normal.pvalue"]]`$). + +The nucleotide diversity of the target samples is $`r display.num(div_values[["diversity"]], 3)`$ +(vertical line in @fig-div). Assuming the context subsets are independent, the +`r div_values[["type.test"]]` test gives a p-value of $`r display.num(div_values[["p.value"]], 3)`$ +for observing a nucleotide diversity as low as that of the target samples. + +![Distribution of nucleotide diversity (π) calculated from $`r div_values[["boot.reps"]]`$ +random subsets of $`r div_values[["sample.size"]]`$ sequences from the context dataset. +The shaded area shows the empirical density; the overlaid curve is the normal distribution +with the same mean and standard deviation. +The vertical line marks the π value of the target samples.](`r params$div`){#fig-div} ## Evolutionary trajectory of the serially-sampled SARS-CoV-2 infection @@ -205,7 +200,7 @@ target samples.](`r params$div`){#fig-div} Sites with minor allele frequency $> `r params$min_ivar_freq`$ are considered polymorphic. The linear association between the collection date of the samples and the number of -polymorphic sites has an $R^2$ of $`r nv.counts[["r2"]]`$ and a p-value of +polymorphic sites has an $R^2$ of $`r display.num(nv.counts[["r2"]], 4)`$ and a p-value of $`r nv.counts[["value"]]`$ (@fig-fig_cor_snp). ![Number of polymorphic sites along time. The @@ -267,7 +262,7 @@ frequency-weighted distances.](`r params$tree`){#fig-tree} To estimate the evolutionary rate, root-to-tip distances measured on the previous tree (@fig-tree) have been correlated with time, obtaining a $R^2$ of -$`r correlation`$ and a p-value of $`r p_value_lm`$. The estimated evolutionary +$`r display.num(correlation, 4)`$ and a p-value of $`r p_value_lm`$. The estimated evolutionary rate is $`r sub_rate`$ number of changes per year (@fig-tempest). ![Scatterplot depicting the relationship between root-to-tip diff --git a/workflow/scripts/report/diversity_plot.R b/workflow/scripts/report/diversity_plot.R index 7118029..0c42118 100644 --- a/workflow/scripts/report/diversity_plot.R +++ b/workflow/scripts/report/diversity_plot.R @@ -31,21 +31,25 @@ p <- data.frame(pi = divs) %>% fill = DIVERSITY_PALETTE["density_fill"], alpha = 0.7, bw = 0.000001, + linewidth = 0.5, color = DIVERSITY_PALETTE["density_color"] ) + - geom_vline( - xintercept = json$diversity, - color = DIVERSITY_PALETTE["value_color"] - ) + stat_function( fun = dnorm, args = list(mean = mean(divs), sd = sd(divs)), - color = DIVERSITY_PALETTE["dnorm_color"] + color = DIVERSITY_PALETTE["dnorm_color"], + linewidth = 1 + ) + + geom_vline( + xintercept = json$diversity, + color = DIVERSITY_PALETTE["value_color"], + linewidth = 1 ) + labs( x = "π", y = "Density" - ) + ) + + xlim(0, NA) ggsave( filename = snakemake@output[["plot"]], From c416300a44b4a3e21c49881d0bdbd85f6f0f4c93 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 11 Nov 2025 15:38:13 +0100 Subject: [PATCH 135/138] Update timestamp format --- template.qmd | 3 +-- 1 file changed, 1 insertion(+), 2 deletions(-) diff --git a/template.qmd b/template.qmd index f8c22cc..522dfad 100644 --- a/template.qmd +++ b/template.qmd @@ -56,7 +56,6 @@ output-file: report.html display.num <- function(n, digits) { format(n, digits = digits, nsmall = digits) } -Sys.setlocale("LC_ALL", "English") ``` ```{r read, echo = F, message = F, include = F} @@ -115,7 +114,7 @@ if (params$use_bionj) { # Freyja timestamp freyja.timestamp <- read_file(params$freyja_ts) %>% as.POSIXct(., format = "%m_%d_%Y-%H-%M") %>% - strftime(., format = "%d %B %Y at %H:%M") + strftime(., format = "%Y-%m-%d at %H:%M") ``` ## Summary of the target samples dataset From 01438bc17046061b66b8115abea07a698eae56fe Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 11 Nov 2025 15:42:02 +0100 Subject: [PATCH 136/138] Update wording --- template.qmd | 8 ++++---- 1 file changed, 4 insertions(+), 4 deletions(-) diff --git a/template.qmd b/template.qmd index 522dfad..3c2d044 100644 --- a/template.qmd +++ b/template.qmd @@ -207,7 +207,7 @@ blue line shows the linear model fit.](`r params$fig_cor_snp`){#fig-fig_cor_snp} ### Description of intra-host nucleotide variants -A total of $`r n_SNV`$ different single nucleotide variants (SNV) and $`r n_INDELS`$ +A total of $`r n_SNV`$ different nucleotide variants (NV) and $`r n_INDELS`$ insertions and deletions (indels) have been detected along the genome (@fig-SNV). ::: {.panel-tabset} @@ -216,7 +216,7 @@ insertions and deletions (indels) have been detected along the genome (@fig-SNV) ![Summary of the intra-host accumulation of nucleotide variants, using the reconstructed dataset ancestor as reference. A) Nucleotide -variants per site along the SARS-CoV-2 genome. Relative abundance of NVs is calculated +variants per site along the SARS-CoV-2 genome. Relative abundance of variants is calculated with a sliding window of width $`r nv.counts[["window"]]`$ nucleotides and a step of $`r nv.counts[["step"]]`$. Labels indicate the coding regions of the non structural proteins (NSP) within ORF1ab. B) Genome variation along the genome for each sample. @@ -227,7 +227,7 @@ at the bottom, and the latest, at the top.](`r params$SNV`){#fig-SNV} ![Summary of the intra-host accumulation of nucleotide variants in the spike sequence, using the reconstructed dataset ancestor as reference. A) Nucleotide -variants per site along the S gene. Relative abundance of NVs is calculated +variants per site along the S gene. Relative abundance of variants is calculated with a sliding window of width $`r nv.counts[["window"]]`$ nucleotides and a step of $`r nv.counts[["step"]]`$. B) Genome variation along the S gene for each sample. The Y-axis displays samples in chronological order, with the earliest collection date @@ -237,7 +237,7 @@ at the bottom, and the latest, at the top.](`r params$SNV_s`){#fig-SNV_s} ### Temporal signal of the intra-host mutations -The correlation of the allele frequency of each NV with the time since the +`r cor.method.name` correlation of the allele frequency of each variant with the time since the initial sampling has been calculated (@fig-volcano). ![`r cor.method.name` correlation coefficients and adjusted p-values of From e0f380f035447c3cecf8523897384e89e82b39de Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Tue, 11 Nov 2025 15:48:29 +0100 Subject: [PATCH 137/138] Update Dockerfile --- Dockerfile | 11 +++++------ 1 file changed, 5 insertions(+), 6 deletions(-) diff --git a/Dockerfile b/Dockerfile index ce29cbd..63810c9 100644 --- a/Dockerfile +++ b/Dockerfile @@ -1,6 +1,6 @@ FROM condaforge/miniforge3:latest LABEL io.github.snakemake.containerized="true" -LABEL io.github.snakemake.conda_env_hash="d883c5236d6779c015257dd3adab712fee0805655dfc5ccdc99b24758b7249a5" +LABEL io.github.snakemake.conda_env_hash="7071b22b1161190c06be3ac061ab0019a1a8d3038532c9134e070a3875414ef5" # Step 2: Retrieve conda environments @@ -99,7 +99,7 @@ COPY workflow/envs/pangolin.yaml /conda-envs/fd645c541ee7a3d43fb9167441b77888/en # Conda environment: # source: workflow/envs/quarto_render.yaml -# prefix: /conda-envs/b33b29b10147e6767116c7a41920d484 +# prefix: /conda-envs/96f3c1cec4b3ce5d72f708992272e9c1 # channels: # - conda-forge # dependencies: @@ -108,10 +108,9 @@ COPY workflow/envs/pangolin.yaml /conda-envs/fd645c541ee7a3d43fb9167441b77888/en # - quarto==1.8.25 # - deno==2.3.1 # - r-tidyverse==2.0.0 -# - r-quarto==1.5.1 # - r-heatmaply==1.6.0 -RUN mkdir -p /conda-envs/b33b29b10147e6767116c7a41920d484 -COPY workflow/envs/quarto_render.yaml /conda-envs/b33b29b10147e6767116c7a41920d484/environment.yaml +RUN mkdir -p /conda-envs/96f3c1cec4b3ce5d72f708992272e9c1 +COPY workflow/envs/quarto_render.yaml /conda-envs/96f3c1cec4b3ce5d72f708992272e9c1/environment.yaml # Conda environment: # source: workflow/envs/renv.yaml @@ -169,7 +168,7 @@ RUN conda env create --prefix /conda-envs/9c24a867826615972cc288081976e7fc --fil conda env create --prefix /conda-envs/0a608afb24723cb6fa8aef748f5efbc8 --file /conda-envs/0a608afb24723cb6fa8aef748f5efbc8/environment.yaml && \ conda env create --prefix /conda-envs/04a3347f94ddf7e21c34bc49e5246076 --file /conda-envs/04a3347f94ddf7e21c34bc49e5246076/environment.yaml && \ conda env create --prefix /conda-envs/fd645c541ee7a3d43fb9167441b77888 --file /conda-envs/fd645c541ee7a3d43fb9167441b77888/environment.yaml && \ - conda env create --prefix /conda-envs/b33b29b10147e6767116c7a41920d484 --file /conda-envs/b33b29b10147e6767116c7a41920d484/environment.yaml && \ + conda env create --prefix /conda-envs/96f3c1cec4b3ce5d72f708992272e9c1 --file /conda-envs/96f3c1cec4b3ce5d72f708992272e9c1/environment.yaml && \ conda env create --prefix /conda-envs/8ad6cdcf265d30289788da99d5bf9fff --file /conda-envs/8ad6cdcf265d30289788da99d5bf9fff/environment.yaml && \ conda env create --prefix /conda-envs/0adafb79cb1bec58ef4c77bf4cca4f95 --file /conda-envs/0adafb79cb1bec58ef4c77bf4cca4f95/environment.yaml && \ conda env create --prefix /conda-envs/5150d0f0a91d7f7a789a06f453d63479 --file /conda-envs/5150d0f0a91d7f7a789a06f453d63479/environment.yaml && \ From d83d05872123fc3425ec21e8477694783c006ede Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Miguel=20=C3=81lvarez=20Herrera?= Date: Thu, 13 Nov 2025 17:23:03 +0100 Subject: [PATCH 138/138] Fix post-deploy installation in Dockerfile --- Dockerfile | 1 + 1 file changed, 1 insertion(+) diff --git a/Dockerfile b/Dockerfile index 63810c9..7f6ac6a 100644 --- a/Dockerfile +++ b/Dockerfile @@ -63,6 +63,7 @@ COPY workflow/envs/freyja.yaml /conda-envs/bb4c5f3a509433cc08861582fab4a705/envi # - r-logger==0.2.2 RUN mkdir -p /conda-envs/3fad3c9cdfa40bee9404f6a2e8fda69f COPY workflow/envs/gisaidr.yaml /conda-envs/3fad3c9cdfa40bee9404f6a2e8fda69f/environment.yaml +COPY workflow/envs/gisaidr.post-deploy.sh /conda-envs/3fad3c9cdfa40bee9404f6a2e8fda69f/post-deploy.sh # Conda environment: # source: workflow/envs/iqtree.yaml