From d60b41fa62a46bef60baadd65f06348c0e16972b Mon Sep 17 00:00:00 2001 From: Felix Krueger Date: Wed, 8 Apr 2026 14:27:55 +0200 Subject: [PATCH 1/7] Filter Bismark coverage files in targeted sequencing mode When run_targeted_sequencing is enabled, only bedGraph files were filtered against the target regions BED file. The Bismark .cov.gz files (used by downstream tools like methylKit, bsseq, and DSS) were published unfiltered, containing off-target CpG sites. This adds a second BEDTOOLS_INTERSECT call (via process alias BEDTOOLS_INTERSECT_COV) to filter .cov files the same way, publishing them alongside the filtered bedGraphs. Only applies to the Bismark aligner path since bwameth/MethylDackel does not produce .cov files. --- conf/modules/bedtools_intersect_cov.config | 14 + conf/subworkflows/targeted_sequencing.config | 1 + .../local/targeted_sequencing/main.nf | 68 ++-- workflows/methylseq/main.nf | 316 +++++++++--------- 4 files changed, 213 insertions(+), 186 deletions(-) create mode 100644 conf/modules/bedtools_intersect_cov.config diff --git a/conf/modules/bedtools_intersect_cov.config b/conf/modules/bedtools_intersect_cov.config new file mode 100644 index 00000000..561925ca --- /dev/null +++ b/conf/modules/bedtools_intersect_cov.config @@ -0,0 +1,14 @@ +process { + withName: BEDTOOLS_INTERSECT_COV { + ext.args = '' + ext.prefix = { "${intervals1.baseName}" } + ext.suffix = 'targeted.cov' + publishDir = [ + [ + path: { "${params.outdir}/bismark/methylation_calls/methylation_coverage" }, + mode: params.publish_dir_mode, + pattern: "*targeted.cov" + ] + ] + } +} diff --git a/conf/subworkflows/targeted_sequencing.config b/conf/subworkflows/targeted_sequencing.config index 666eb0d7..3bb996ad 100644 --- a/conf/subworkflows/targeted_sequencing.config +++ b/conf/subworkflows/targeted_sequencing.config @@ -1,4 +1,5 @@ includeConfig "../modules/bedtools_intersect.config" +includeConfig "../modules/bedtools_intersect_cov.config" includeConfig "../modules/picard_bedtointervallist.config" includeConfig "../modules/picard_createsequencedictionary.config" includeConfig "../modules/picard_collecthsmetrics.config" diff --git a/subworkflows/local/targeted_sequencing/main.nf b/subworkflows/local/targeted_sequencing/main.nf index f9d20aa9..472326f8 100644 --- a/subworkflows/local/targeted_sequencing/main.nf +++ b/subworkflows/local/targeted_sequencing/main.nf @@ -1,27 +1,28 @@ /* * targeted_sequencing subworkflow * - * Filters bedGraph files with the target_regions BED file so they contain positions only listed in the BED. + * Filters bedGraph and coverage files with the target_regions BED file so they contain positions only listed in the BED. * If specified, it also generates some performance metrics with Picard CollectHsMetrics, such as Fold-80 Base Penalty, * HS Library Size, Percent Duplicates, and Percent Off Bait. This is relevant for methylome experiments with targeted seq. */ -include { BEDTOOLS_INTERSECT } from '../../../modules/nf-core/bedtools/intersect/main' -include { PICARD_CREATESEQUENCEDICTIONARY } from '../../../modules/nf-core/picard/createsequencedictionary/main' -include { PICARD_BEDTOINTERVALLIST } from '../../../modules/nf-core/picard/bedtointervallist/main' -include { PICARD_COLLECTHSMETRICS } from '../../../modules/nf-core/picard/collecthsmetrics/main' +include { BEDTOOLS_INTERSECT } from '../../../modules/nf-core/bedtools/intersect/main' +include { BEDTOOLS_INTERSECT as BEDTOOLS_INTERSECT_COV } from '../../../modules/nf-core/bedtools/intersect/main' +include { PICARD_CREATESEQUENCEDICTIONARY } from '../../../modules/nf-core/picard/createsequencedictionary/main' +include { PICARD_BEDTOINTERVALLIST } from '../../../modules/nf-core/picard/bedtointervallist/main' +include { PICARD_COLLECTHSMETRICS } from '../../../modules/nf-core/picard/collecthsmetrics/main' workflow TARGETED_SEQUENCING { - take: - ch_bedgraph // channel: [ val(meta), [ bedGraph(s) ]] when bwameth, [ val(meta), bedGraph ] when bismark - ch_target_regions // channel: path(target_regions.bed) - ch_fasta // channel: [ [:], /path/to/genome.fa] - ch_fasta_index // channel: [ val(meta), /path/to/genome.fa.fai] - ch_bam // channel: [ val(meta), [ bam ] ] ## BAM from alignment - ch_bai // channel: [ val(meta), [ bai ] ] ## BAI from alignment - ch_gzi // channel: [ val(meta), [ gzi ] ] ## GZI from fasta - collecthsmetrics // boolean: whether to run Picard CollectHsMetrics + ch_bedgraph // channel: [ val(meta), [ bedGraph(s) ]] when bwameth, [ val(meta), bedGraph ] when bismark + ch_coverage // channel: [ val(meta), cov.gz ] from Bismark methylation extractor (empty for bwameth) + ch_target_regions // channel: path(target_regions.bed) + ch_fasta // channel: [ [:], /path/to/genome.fa] + ch_fasta_index // channel: [ val(meta), /path/to/genome.fa.fai] + ch_bam // channel: [ val(meta), [ bam ] ] ## BAM from alignment + ch_bai // channel: [ val(meta), [ bai ] ] ## BAI from alignment + ch_gzi // channel: [ val(meta), [ gzi ] ] ## GZI from fasta + collecthsmetrics // boolean: whether to run Picard CollectHsMetrics main: @@ -39,10 +40,23 @@ workflow TARGETED_SEQUENCING { BEDTOOLS_INTERSECT( ch_bedgraphs_target, - [[:], []] + [[:], []], ) ch_versions = ch_versions.mix(BEDTOOLS_INTERSECT.out.versions) + /* + * Intersect Bismark coverage files with target regions + * The .cov.gz files are filtered the same way as bedGraph files so that + * downstream tools (methylKit, bsseq, DSS) receive only on-target CpGs. + */ + ch_coverage_target = ch_coverage.combine(ch_target_regions) + + BEDTOOLS_INTERSECT_COV( + ch_coverage_target, + [[:], []], + ) + ch_versions = ch_versions.mix(BEDTOOLS_INTERSECT_COV.out.versions) + /* * Run Picard CollectHSMetrics */ @@ -65,7 +79,7 @@ workflow TARGETED_SEQUENCING { PICARD_BEDTOINTERVALLIST( target_regions_with_meta, ch_sequence_dictionary, - [] + [], ) ch_intervals = PICARD_BEDTOINTERVALLIST.out.intervallist.map { it[1] } ch_versions = ch_versions.mix(PICARD_BEDTOINTERVALLIST.out.versions) @@ -75,7 +89,8 @@ workflow TARGETED_SEQUENCING { * Note: Using the same intervals for both target and bait as they are typically * the same for targeted methylation sequencing experiments */ - ch_picard_inputs = ch_bam.join(ch_bai) + ch_picard_inputs = ch_bam + .join(ch_bai) .combine(ch_intervals) .combine(ch_intervals) .combine(ch_fasta) @@ -83,11 +98,11 @@ workflow TARGETED_SEQUENCING { .combine(ch_sequence_dictionary) .combine(ch_gzi) .multiMap { meta, bam, bai, intervals1, intervals2, meta_fasta, fasta, meta_fasta_index, fasta_index, meta_dict, dict, meta_gzi, gzi -> - bam_etc: [ meta, bam, bai, intervals1, intervals2 ] // intervals: baits, targets - fasta: [ meta_fasta, fasta ] - fasta_index: [ meta_fasta_index, fasta_index ] - dict: [ meta_dict, dict ] - gzi : [ meta_gzi, gzi ] + bam_etc: [meta, bam, bai, intervals1, intervals2] + fasta: [meta_fasta, fasta] + fasta_index: [meta_fasta_index, fasta_index] + dict: [meta_dict, dict] + gzi: [meta_gzi, gzi] } PICARD_COLLECTHSMETRICS( @@ -95,14 +110,15 @@ workflow TARGETED_SEQUENCING { ch_picard_inputs.fasta, ch_picard_inputs.fasta_index, ch_picard_inputs.dict, - ch_picard_inputs.gzi + ch_picard_inputs.gzi, ) ch_picard_metrics = PICARD_COLLECTHSMETRICS.out.metrics ch_versions = ch_versions.mix(PICARD_COLLECTHSMETRICS.out.versions) } emit: - bedgraph_filtered = BEDTOOLS_INTERSECT.out.intersect // channel: [ val(meta), path("*.bedGraph") ] - picard_metrics = ch_picard_metrics // channel: [ val(meta), path("*_metrics") ] - versions = ch_versions // channel: path("*.version.txt") + bedgraph_filtered = BEDTOOLS_INTERSECT.out.intersect // channel: [ val(meta), path("*.bedGraph") ] + coverage_filtered = BEDTOOLS_INTERSECT_COV.out.intersect // channel: [ val(meta), path("*.cov") ] + picard_metrics = ch_picard_metrics // channel: [ val(meta), path("*_metrics") ] + versions = ch_versions // channel: path("*.version.txt") } diff --git a/workflows/methylseq/main.nf b/workflows/methylseq/main.nf index 9630375d..b4d28e01 100644 --- a/workflows/methylseq/main.nf +++ b/workflows/methylseq/main.nf @@ -32,66 +32,66 @@ include { TARGETED_SEQUENCING } from '../../subworkflows/local/targete */ workflow METHYLSEQ { - take: - samplesheet // channel: [ path(samplesheet.csv) ] - ch_versions // channel: [ path(versions.yml) ] - ch_fasta // channel: [ path(fasta) ] - ch_fasta_index // channel: [ path(fasta index) ] - ch_bismark_index // channel: [ path(bismark index) ] - ch_bwameth_index // channel: [ path(bwameth index) ] - ch_bwamem_index // channel: [ path(bwamem_index) ] + samplesheet // channel: [ path(samplesheet.csv) ] + ch_versions // channel: [ path(versions.yml) ] + ch_fasta // channel: [ path(fasta) ] + ch_fasta_index // channel: [ path(fasta index) ] + ch_bismark_index // channel: [ path(bismark index) ] + ch_bwameth_index // channel: [ path(bwameth index) ] + ch_bwamem_index // channel: [ path(bwamem_index) ] main: - ch_fastq = channel.empty() - ch_fastqc_html = channel.empty() - ch_fastqc_zip = channel.empty() - ch_reads = channel.empty() - ch_bam = channel.empty() - ch_bai = channel.empty() - ch_gzi = channel.empty() - ch_bedgraph = channel.empty() - ch_aligner_mqc = channel.empty() + ch_fastq = channel.empty() + ch_fastqc_html = channel.empty() + ch_fastqc_zip = channel.empty() + ch_reads = channel.empty() + ch_bam = channel.empty() + ch_bai = channel.empty() + ch_gzi = channel.empty() + ch_bedgraph = channel.empty() + ch_coverage = channel.empty() + ch_aligner_mqc = channel.empty() ch_rastair_mbias = channel.empty() - ch_rastair_call = channel.empty() - ch_methylkit = channel.empty() - ch_mbias = channel.empty() - ch_qualimap = channel.empty() - ch_preseq = channel.empty() + ch_rastair_call = channel.empty() + ch_methylkit = channel.empty() + ch_mbias = channel.empty() + ch_qualimap = channel.empty() + ch_preseq = channel.empty() ch_multiqc_files = channel.empty() // // Branch channels from input samplesheet channel // - ch_samplesheet = samplesheet - .branch { meta, fastqs -> - single : fastqs.size() == 1 - return [ meta, fastqs.flatten() ] - multiple: fastqs.size() > 1 - return [ meta, fastqs.flatten() ] - } + ch_samplesheet = samplesheet.branch { meta, fastqs -> + single: fastqs.size() == 1 + return [meta, fastqs.flatten()] + multiple: fastqs.size() > 1 + return [meta, fastqs.flatten()] + } // // MODULE: Concatenate FastQ files from same sample if required // - CAT_FASTQ ( + CAT_FASTQ( ch_samplesheet.multiple ) - ch_fastq = CAT_FASTQ.out.reads.mix(ch_samplesheet.single) + ch_fastq = CAT_FASTQ.out.reads.mix(ch_samplesheet.single) ch_versions = ch_versions.mix(CAT_FASTQ.out.versions) // // MODULE: Run FastQC // if (!params.skip_fastqc) { - FASTQC ( + FASTQC( ch_fastq ) - ch_fastqc_html = FASTQC.out.html - ch_fastqc_zip = FASTQC.out.zip - } else { - ch_fastqc_html = channel.empty() - ch_fastqc_zip = channel.empty() + ch_fastqc_html = FASTQC.out.html + ch_fastqc_zip = FASTQC.out.zip + } + else { + ch_fastqc_html = channel.empty() + ch_fastqc_zip = channel.empty() } // @@ -101,10 +101,11 @@ workflow METHYLSEQ { TRIMGALORE( ch_fastq ) - ch_reads = TRIMGALORE.out.reads + ch_reads = TRIMGALORE.out.reads ch_versions = ch_versions.mix(TRIMGALORE.out.versions) - } else { - ch_reads = ch_fastq + } + else { + ch_reads = ch_fastq } // @@ -112,11 +113,11 @@ workflow METHYLSEQ { // if (params.taps && params.aligner != 'bwamem') { - log.info "TAPS protocol detected and aligner is not 'bwamem'. We recommend using bwa-mem for TAPS protocol as it is optimized for this type of data." + log.info("TAPS protocol detected and aligner is not 'bwamem'. We recommend using bwa-mem for TAPS protocol as it is optimized for this type of data.") } // Aligner: bismark or bismark_hisat - if (params.aligner =~ /bismark/ ) { + if (params.aligner =~ /bismark/) { // // Run Bismark alignment + downstream processing // @@ -124,82 +125,79 @@ workflow METHYLSEQ { .combine(ch_fasta) .combine(ch_bismark_index) .multiMap { meta, reads, meta_fasta, fasta, meta_bismark, bismark_index -> - reads: [ meta, reads ] - fasta: [ meta_fasta, fasta ] - bismark_index: [ meta_bismark, bismark_index ] + reads: [meta, reads] + fasta: [meta_fasta, fasta] + bismark_index: [meta_bismark, bismark_index] } - FASTQ_ALIGN_DEDUP_BISMARK ( + FASTQ_ALIGN_DEDUP_BISMARK( ch_bismark_inputs.reads, ch_bismark_inputs.fasta, ch_bismark_inputs.bismark_index, params.skip_deduplication || params.rrbs, - params.cytosine_report || params.nomeseq + params.cytosine_report || params.nomeseq, ) - ch_bam = FASTQ_ALIGN_DEDUP_BISMARK.out.bam - ch_bai = FASTQ_ALIGN_DEDUP_BISMARK.out.bai - ch_bedgraph = FASTQ_ALIGN_DEDUP_BISMARK.out.methylation_bedgraph + ch_bam = FASTQ_ALIGN_DEDUP_BISMARK.out.bam + ch_bai = FASTQ_ALIGN_DEDUP_BISMARK.out.bai + ch_bedgraph = FASTQ_ALIGN_DEDUP_BISMARK.out.methylation_bedgraph + ch_coverage = FASTQ_ALIGN_DEDUP_BISMARK.out.methylation_coverage ch_aligner_mqc = FASTQ_ALIGN_DEDUP_BISMARK.out.multiqc - ch_versions = ch_versions.mix(FASTQ_ALIGN_DEDUP_BISMARK.out.versions) + ch_versions = ch_versions.mix(FASTQ_ALIGN_DEDUP_BISMARK.out.versions) } - // Aligner: bwameth - else if (params.aligner == 'bwameth'){ + else if (params.aligner == 'bwameth') { ch_bwameth_inputs = ch_reads .combine(ch_fasta) .combine(ch_fasta_index) .combine(ch_bwameth_index) .multiMap { meta, reads, meta_fasta, fasta, meta_fasta_index, fasta_index, meta_bwameth, bwameth_index -> - reads: [ meta, reads ] - fasta: [ meta_fasta, fasta ] - fasta_index: [ meta_fasta_index, fasta_index ] - bwameth_index: [ meta_bwameth, bwameth_index ] + reads: [meta, reads] + fasta: [meta_fasta, fasta] + fasta_index: [meta_fasta_index, fasta_index] + bwameth_index: [meta_bwameth, bwameth_index] } - FASTQ_ALIGN_DEDUP_BWAMETH ( + FASTQ_ALIGN_DEDUP_BWAMETH( ch_bwameth_inputs.reads, ch_bwameth_inputs.fasta, ch_bwameth_inputs.fasta_index, ch_bwameth_inputs.bwameth_index, params.skip_deduplication || params.rrbs, - workflow.profile.tokenize(',').intersect(['gpu']).size() >= 1 + workflow.profile.tokenize(',').intersect(['gpu']).size() >= 1, ) - ch_bam = FASTQ_ALIGN_DEDUP_BWAMETH.out.bam - ch_bai = FASTQ_ALIGN_DEDUP_BWAMETH.out.bai + ch_bam = FASTQ_ALIGN_DEDUP_BWAMETH.out.bam + ch_bai = FASTQ_ALIGN_DEDUP_BWAMETH.out.bai ch_aligner_mqc = FASTQ_ALIGN_DEDUP_BWAMETH.out.multiqc - ch_versions = ch_versions.mix(FASTQ_ALIGN_DEDUP_BWAMETH.out.versions) + ch_versions = ch_versions.mix(FASTQ_ALIGN_DEDUP_BWAMETH.out.versions) } - - // Aligner: bwamem - else if (params.aligner == 'bwamem'){ + else if (params.aligner == 'bwamem') { ch_bwamem_inputs = ch_reads .combine(ch_fasta) .combine(ch_fasta_index) .combine(ch_bwamem_index) .multiMap { meta, reads, meta_fasta, fasta, meta_fasta_index, fasta_index, meta_bwamem, bwamem_index -> - reads: [ meta, reads ] - fasta: [ meta_fasta, fasta ] - fasta_index: [ meta_fasta_index, fasta_index ] - bwamem_index: [ meta_bwamem, bwamem_index ] + reads: [meta, reads] + fasta: [meta_fasta, fasta] + fasta_index: [meta_fasta_index, fasta_index] + bwamem_index: [meta_bwamem, bwamem_index] } - FASTQ_ALIGN_DEDUP_BWAMEM ( + FASTQ_ALIGN_DEDUP_BWAMEM( ch_bwamem_inputs.reads, ch_bwamem_inputs.fasta, ch_bwamem_inputs.fasta_index, ch_bwamem_inputs.bwamem_index, - params.skip_deduplication + params.skip_deduplication, ) - ch_bam = FASTQ_ALIGN_DEDUP_BWAMEM.out.bam - ch_bai = FASTQ_ALIGN_DEDUP_BWAMEM.out.bai + ch_bam = FASTQ_ALIGN_DEDUP_BWAMEM.out.bam + ch_bai = FASTQ_ALIGN_DEDUP_BWAMEM.out.bai ch_aligner_mqc = FASTQ_ALIGN_DEDUP_BWAMEM.out.multiqc - ch_versions = ch_versions.mix(FASTQ_ALIGN_DEDUP_BWAMEM.out.versions.unique{ it.baseName }) + ch_versions = ch_versions.mix(FASTQ_ALIGN_DEDUP_BWAMEM.out.versions.unique { it.baseName }) } - else { - error "ERROR: Invalid aligner '${params.aligner}'. Valid options are: 'bismark', 'bismark_hisat', 'bwameth' or 'bwamem'." + error("ERROR: Invalid aligner '${params.aligner}'. Valid options are: 'bismark', 'bismark_hisat', 'bwameth' or 'bwamem'.") } // @@ -209,62 +207,63 @@ workflow METHYLSEQ { ch_bam_bai = ch_bam.join(ch_bai) ch_taps_inputs = ch_bam_bai - .combine(ch_fasta) // broadcast fasta - .combine(ch_fasta_index) // broadcast fai + .combine(ch_fasta) + .combine(ch_fasta_index) .multiMap { meta, bam, bai, _meta_fasta, fasta, _meta_fai, fai -> - bam: [ meta, bam ] // use sample meta so subworkflow aligns properly - bai: [ meta, bai ] // use sample meta so subworkflow aligns properly - fasta: [ meta, fasta ] // use sample meta so subworkflow aligns properly - fasta_index: [ meta, fai ] // same here + bam: [meta, bam] + bai: [meta, bai] + fasta: [meta, fasta] + fasta_index: [meta, fai] } BAM_TAPS_CONVERSION( ch_taps_inputs.bam, ch_taps_inputs.bai, ch_taps_inputs.fasta, - ch_taps_inputs.fasta_index + ch_taps_inputs.fasta_index, ) - ch_rastair_mbias = BAM_TAPS_CONVERSION.out.mbias // channel: [ val(meta), [ txt ] ] - ch_rastair_call = BAM_TAPS_CONVERSION.out.call // channel: [ val(meta), [ txt ] ] - ch_versions = ch_versions.mix(BAM_TAPS_CONVERSION.out.versions) + ch_rastair_mbias = BAM_TAPS_CONVERSION.out.mbias + // channel: [ val(meta), [ txt ] ] + ch_rastair_call = BAM_TAPS_CONVERSION.out.call + // channel: [ val(meta), [ txt ] ] + ch_versions = ch_versions.mix(BAM_TAPS_CONVERSION.out.versions) } - - // - // Subworkflow: Count negative C->T conversion rates as a readout for DNA methylation - // else if (!params.taps && params.aligner == 'bwameth') { ch_bam_bai = ch_bam.join(ch_bai) ch_methyldackel_inputs = ch_bam_bai - .combine(ch_fasta) // broadcast fasta - .combine(ch_fasta_index) // broadcast fai + .combine(ch_fasta) + .combine(ch_fasta_index) .multiMap { meta, bam, bai, _meta_fasta, fasta, _meta_fai, fai -> - bam: [ meta, bam ] // use sample meta so subworkflow aligns properly - bai: [ meta, bai ] // use sample meta so subworkflow aligns properly - fasta: [ meta, fasta ] // use sample meta so subworkflow aligns properly - fasta_index: [ meta, fai ] // same here + bam: [meta, bam] + bai: [meta, bai] + fasta: [meta, fasta] + fasta_index: [meta, fai] } - BAM_METHYLDACKEL ( + BAM_METHYLDACKEL( ch_methyldackel_inputs.bam, ch_methyldackel_inputs.bai, ch_methyldackel_inputs.fasta, - ch_methyldackel_inputs.fasta_index + ch_methyldackel_inputs.fasta_index, ) - ch_bedgraph = BAM_METHYLDACKEL.out.methydackel_extract_bedgraph // channel: [ val(meta), [ bedgraph ] ] - ch_methylkit = BAM_METHYLDACKEL.out.methydackel_extract_methylkit // channel: [ val(meta), [ methylkit ] ] - ch_mbias = BAM_METHYLDACKEL.out.methydackel_mbias // channel: [ val(meta), [ mbias ] ] - ch_versions = ch_versions.mix(BAM_METHYLDACKEL.out.versions) + ch_bedgraph = BAM_METHYLDACKEL.out.methydackel_extract_bedgraph + // channel: [ val(meta), [ bedgraph ] ] + ch_methylkit = BAM_METHYLDACKEL.out.methydackel_extract_methylkit + // channel: [ val(meta), [ methylkit ] ] + ch_mbias = BAM_METHYLDACKEL.out.methydackel_mbias + // channel: [ val(meta), [ mbias ] ] + ch_versions = ch_versions.mix(BAM_METHYLDACKEL.out.versions) } // // MODULE: Qualimap BamQC // skipped by default. to use run with `--run_qualimap` param. // - if(params.run_qualimap) { - QUALIMAP_BAMQC ( + if (params.run_qualimap) { + QUALIMAP_BAMQC( ch_bam, - params.bamqc_regions_file ? channel.fromPath( params.bamqc_regions_file, checkIfExists: true ).toList() : [] + params.bamqc_regions_file ? channel.fromPath(params.bamqc_regions_file, checkIfExists: true).toList() : [], ) ch_qualimap = QUALIMAP_BAMQC.out.results ch_versions = ch_versions.mix(QUALIMAP_BAMQC.out.versions) @@ -274,22 +273,23 @@ workflow METHYLSEQ { // MODULE: Targeted sequencing analysis // skipped by default. to use run with `--run_targeted_sequencing` param. // - if (params.run_targeted_sequencing){ + if (params.run_targeted_sequencing) { if (params.taps || params.aligner == 'bwamem') { - error "ERROR: --run_targeted_sequencing can't be running using rastair (methylation caller for TAPS) " + error("ERROR: --run_targeted_sequencing can't be running using rastair (methylation caller for TAPS) ") } if (!params.target_regions_file) { - error "ERROR: --target_regions_file must be specified when using --run_targeted_sequencing" + error("ERROR: --target_regions_file must be specified when using --run_targeted_sequencing") } - TARGETED_SEQUENCING ( + TARGETED_SEQUENCING( ch_bedgraph, + ch_coverage, channel.fromPath(params.target_regions_file, checkIfExists: true), ch_fasta, ch_fasta_index, ch_bam, ch_bai, ch_gzi, - params.collecthsmetrics + params.collecthsmetrics, ) ch_versions = ch_versions.mix(TARGETED_SEQUENCING.out.versions) } @@ -298,11 +298,11 @@ workflow METHYLSEQ { // MODULE: Preseq LCEXTRAP // skipped by default. to use run with `--run_preseq` param. // - if(params.run_preseq) { - PRESEQ_LCEXTRAP ( + if (params.run_preseq) { + PRESEQ_LCEXTRAP( ch_bam ) - ch_preseq = PRESEQ_LCEXTRAP.out.lc_extrap + ch_preseq = PRESEQ_LCEXTRAP.out.lc_extrap ch_versions = ch_versions.mix(PRESEQ_LCEXTRAP.out.versions) } @@ -312,10 +312,11 @@ workflow METHYLSEQ { softwareVersionsToYAML(ch_versions) .collectFile( storeDir: "${params.outdir}/pipeline_info", - name: 'nf_core_' + 'methylseq_software_' + 'mqc_' + 'versions.yml', + name: 'nf_core_' + 'methylseq_software_' + 'mqc_' + 'versions.yml', sort: true, - newLine: true - ).set { ch_collated_versions } + newLine: true, + ) + .set { ch_collated_versions } // // Topic channel versions - written separately to avoid blocking MULTIQC @@ -325,93 +326,88 @@ workflow METHYLSEQ { .distinct() .filter { entry -> !(entry instanceof Path) } .map { process, tool, version -> - def processName = process[process.lastIndexOf(':')+1..-1] + def processName = process[process.lastIndexOf(':') + 1..-1] "${processName}:\n ${tool}: ${version}" } .collectFile( storeDir: "${params.outdir}/pipeline_info", name: 'nf_core_methylseq_topic_versions.yml', sort: true, - newLine: true + newLine: true, ) // // MODULE: MultiQC // if (!params.skip_multiqc) { - ch_multiqc_config = channel.fromPath("$projectDir/assets/multiqc_config.yml", checkIfExists: true) - ch_multiqc_custom_config = params.multiqc_config ? - channel.fromPath(params.multiqc_config, checkIfExists: true) : - channel.empty() - ch_multiqc_logo = params.multiqc_logo ? - channel.fromPath(params.multiqc_logo, checkIfExists: true) : - channel.empty() - - summary_params = paramsSummaryMap(workflow, parameters_schema: "nextflow_schema.json") - ch_workflow_summary = channel.value(paramsSummaryMultiqc(summary_params)) - - ch_multiqc_custom_methods_description = params.multiqc_methods_description ? - file(params.multiqc_methods_description, checkIfExists: true) : - file("$projectDir/assets/methods_description_template.yml", checkIfExists: true) - ch_methods_description = channel.value(methodsDescriptionText(ch_multiqc_custom_methods_description)) + ch_multiqc_config = channel.fromPath("${projectDir}/assets/multiqc_config.yml", checkIfExists: true) + ch_multiqc_custom_config = params.multiqc_config + ? channel.fromPath(params.multiqc_config, checkIfExists: true) + : channel.empty() + ch_multiqc_logo = params.multiqc_logo + ? channel.fromPath(params.multiqc_logo, checkIfExists: true) + : channel.empty() + + summary_params = paramsSummaryMap(workflow, parameters_schema: "nextflow_schema.json") + ch_workflow_summary = channel.value(paramsSummaryMultiqc(summary_params)) + + ch_multiqc_custom_methods_description = params.multiqc_methods_description + ? file(params.multiqc_methods_description, checkIfExists: true) + : file("${projectDir}/assets/methods_description_template.yml", checkIfExists: true) + ch_methods_description = channel.value(methodsDescriptionText(ch_multiqc_custom_methods_description)) ch_multiqc_files = ch_multiqc_files.mix(ch_workflow_summary.collectFile(name: 'workflow_summary_mqc.yaml')) ch_multiqc_files = ch_multiqc_files.mix(ch_collated_versions) ch_multiqc_files = ch_multiqc_files.mix( ch_methods_description.collectFile( name: 'methods_description_mqc.yaml', - sort: true + sort: true, ) ) - if(params.run_qualimap) { - ch_multiqc_files = ch_multiqc_files.mix(QUALIMAP_BAMQC.out.results.collect{ it[1] }.ifEmpty([])) + if (params.run_qualimap) { + ch_multiqc_files = ch_multiqc_files.mix(QUALIMAP_BAMQC.out.results.collect { it[1] }.ifEmpty([])) } if (params.run_preseq) { - ch_multiqc_files = ch_multiqc_files.mix(PRESEQ_LCEXTRAP.out.log.collect{ it[1] }.ifEmpty([])) + ch_multiqc_files = ch_multiqc_files.mix(PRESEQ_LCEXTRAP.out.log.collect { it[1] }.ifEmpty([])) } ch_multiqc_files = ch_multiqc_files.mix(ch_aligner_mqc.ifEmpty([])) if (!params.skip_trimming) { - ch_multiqc_files = ch_multiqc_files.mix(TRIMGALORE.out.log.collect{ it[1] }) + ch_multiqc_files = ch_multiqc_files.mix(TRIMGALORE.out.log.collect { it[1] }) } if (params.run_targeted_sequencing) { if (params.collecthsmetrics) { - ch_multiqc_files = ch_multiqc_files.mix(TARGETED_SEQUENCING.out.picard_metrics.collect{ it[1] }.ifEmpty([])) + ch_multiqc_files = ch_multiqc_files.mix(TARGETED_SEQUENCING.out.picard_metrics.collect { it[1] }.ifEmpty([])) } } if (!params.skip_fastqc) { - ch_multiqc_files = ch_multiqc_files.mix(FASTQC.out.zip.collect{ it[1] }.ifEmpty([])) + ch_multiqc_files = ch_multiqc_files.mix(FASTQC.out.zip.collect { it[1] }.ifEmpty([])) } - MULTIQC ( + MULTIQC( ch_multiqc_files.collect(), ch_multiqc_config.toList(), ch_multiqc_custom_config.toList(), ch_multiqc_logo.toList(), [], - [] + [], ) ch_multiqc_report = MULTIQC.out.report.toList() - } else { + } + else { ch_multiqc_report = channel.empty() } emit: - bam = ch_bam // channel: [ val(meta), path(bam) ] - bai = ch_bai // channel: [ val(meta), path(bai) ] - rastair_mbias = ch_rastair_mbias // channel: [ val(meta), path(rastair_mbias) ] - rastair_call = ch_rastair_call // channel: [ val(meta), path(rastair_call) ] - methylkit = ch_methylkit // channel: [ val(meta), path(methylkit) ] - mbias = ch_mbias // channel: [ val(meta), path(mbias) ] - bedgraph = ch_bedgraph // channel: [ val(meta), path(bedgraph) ] - qualimap = ch_qualimap // channel: [ val(meta), path(qualimap) ] - preseq = ch_preseq // channel: [ val(meta), path(preseq) ] - multiqc_report = ch_multiqc_report // channel: [ path(multiqc_report.html ) ] - versions = ch_versions // channel: [ path(versions.yml) ] + bam = ch_bam // channel: [ val(meta), path(bam) ] + bai = ch_bai // channel: [ val(meta), path(bai) ] + rastair_mbias = ch_rastair_mbias // channel: [ val(meta), path(rastair_mbias) ] + rastair_call = ch_rastair_call // channel: [ val(meta), path(rastair_call) ] + methylkit = ch_methylkit // channel: [ val(meta), path(methylkit) ] + mbias = ch_mbias // channel: [ val(meta), path(mbias) ] + bedgraph = ch_bedgraph // channel: [ val(meta), path(bedgraph) ] + qualimap = ch_qualimap // channel: [ val(meta), path(qualimap) ] + preseq = ch_preseq // channel: [ val(meta), path(preseq) ] + multiqc_report = ch_multiqc_report // channel: [ path(multiqc_report.html ) ] + versions = ch_versions // channel: [ path(versions.yml) ] } - -/* -~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ - THE END -~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ -*/ From cbc34961ad8ab97fce64feda1f16a768ed390807 Mon Sep 17 00:00:00 2001 From: Felix Krueger Date: Thu, 9 Apr 2026 10:05:13 +0200 Subject: [PATCH 2/7] Fix CpG-aware boundary handling for bedGraph target filtering MIME-Version: 1.0 Content-Type: text/plain; charset=UTF-8 Content-Transfer-Encoding: 8bit Bismark bedGraph files use 0-based half-open coordinates for single-C positions (e.g., chr1 10788 10789). When a CpG straddles a target boundary — the C falls just outside, the G just inside — a naive bedtools intersect drops it because [10788, 10789) does not overlap a target starting at 10789. Replace the generic BEDTOOLS_INTERSECT call with a local FILTER_BEDGRAPH_TARGETS process that: 1. Extends each bedGraph entry by 1 bp on the right (+1 to end coordinate) to represent the full CpG dinucleotide 2. Runs bedtools intersect against the target regions 3. Restores the original single-base coordinates This ensures bedGraph and coverage filtered outputs contain the same set of on-target CpGs. --- conf/modules/bedtools_intersect.config | 6 +- modules/local/filter_bedgraph_targets.nf | 56 +++++++++++++++++++ .../local/targeted_sequencing/main.nf | 17 +++--- 3 files changed, 66 insertions(+), 13 deletions(-) create mode 100644 modules/local/filter_bedgraph_targets.nf diff --git a/conf/modules/bedtools_intersect.config b/conf/modules/bedtools_intersect.config index 629d072b..46f881fb 100644 --- a/conf/modules/bedtools_intersect.config +++ b/conf/modules/bedtools_intersect.config @@ -1,8 +1,6 @@ process { - withName: BEDTOOLS_INTERSECT { - ext.args = '' - ext.prefix = { "${intervals1.baseName}" } - ext.suffix = 'targeted.bedGraph' + withName: FILTER_BEDGRAPH_TARGETS { + ext.prefix = { "${bedgraph.baseName}" } publishDir = [ [ path: { params.aligner == 'bismark' ? "${params.outdir}/bismark/methylation_calls/bedGraph" : "${params.outdir}/methyldackel" }, diff --git a/modules/local/filter_bedgraph_targets.nf b/modules/local/filter_bedgraph_targets.nf new file mode 100644 index 00000000..a559b649 --- /dev/null +++ b/modules/local/filter_bedgraph_targets.nf @@ -0,0 +1,56 @@ +/* + * Filter bedGraph files against target regions with CpG-aware boundary handling. + * + * Bismark bedGraph files use 0-based half-open coordinates for single-C positions + * (e.g., chr1 10788 10789 for a CpG at 1-based position 10789). When a CpG + * straddles a target boundary — C just outside, G just inside — a naive + * intersection drops it because the single-C interval [10788, 10789) does not + * overlap the target [10789, …). + * + * This process extends each bedGraph entry by 1 bp on the right to represent the + * full CpG dinucleotide, intersects with the target BED, then restores the + * original single-base coordinates. + */ +process FILTER_BEDGRAPH_TARGETS { + tag "${meta.id}" + label 'process_single' + + conda "bioconda::bedtools=2.31.1" + container "${workflow.containerEngine == 'singularity' && !task.ext.singularity_pull_docker_container + ? 'https://depot.galaxyproject.org/singularity/bedtools:2.31.1--hf5e1c6e_0' + : 'biocontainers/bedtools:2.31.1--hf5e1c6e_0'}" + + input: + tuple val(meta), path(bedgraph), path(targets) + + output: + tuple val(meta), path("*.targeted.bedGraph"), emit: intersect + path "versions.yml", emit: versions + + when: + task.ext.when == null || task.ext.when + + script: + def prefix = task.ext.prefix ?: "${meta.id}" + """ + # Extend end coordinate by 1 bp so the interval covers the full CpG dinucleotide + awk 'BEGIN{OFS="\\t"} {\$3=\$3+1; print}' ${bedgraph} > extended.bedGraph + + # Intersect with target regions (-wa: write original -a entry; -u: unique hits only) + bedtools intersect \\ + -a extended.bedGraph \\ + -b ${targets} \\ + -wa \\ + -u \\ + > intersected.bedGraph + + # Restore original single-base end coordinates + awk 'BEGIN{OFS="\\t"} {\$3=\$3-1; print}' intersected.bedGraph \\ + > ${prefix}.targeted.bedGraph + + cat <<-END_VERSIONS > versions.yml + "${task.process}": + bedtools: \$(bedtools --version | sed -e "s/bedtools v//g") + END_VERSIONS + """ +} diff --git a/subworkflows/local/targeted_sequencing/main.nf b/subworkflows/local/targeted_sequencing/main.nf index 472326f8..d0dd9bc9 100644 --- a/subworkflows/local/targeted_sequencing/main.nf +++ b/subworkflows/local/targeted_sequencing/main.nf @@ -6,7 +6,7 @@ * HS Library Size, Percent Duplicates, and Percent Off Bait. This is relevant for methylome experiments with targeted seq. */ -include { BEDTOOLS_INTERSECT } from '../../../modules/nf-core/bedtools/intersect/main' +include { FILTER_BEDGRAPH_TARGETS } from '../../../modules/local/filter_bedgraph_targets' include { BEDTOOLS_INTERSECT as BEDTOOLS_INTERSECT_COV } from '../../../modules/nf-core/bedtools/intersect/main' include { PICARD_CREATESEQUENCEDICTIONARY } from '../../../modules/nf-core/picard/createsequencedictionary/main' include { PICARD_BEDTOINTERVALLIST } from '../../../modules/nf-core/picard/bedtointervallist/main' @@ -30,19 +30,18 @@ workflow TARGETED_SEQUENCING { ch_picard_metrics = Channel.empty() /* - * Intersect bedGraph files with target regions - * Ensure ch_bedgraph contains the bedGraph file(s) in an array and split into individual bedGraphs + * Intersect bedGraph files with target regions (CpG-aware boundary handling) + * Ensure ch_bedgraph contains the bedGraph file(s) in an array and split into individual bedGraphs. + * The FILTER_BEDGRAPH_TARGETS process extends single-C intervals by 1 bp before + * intersection so that CpGs straddling a target boundary are not lost. */ ch_bedgraphs_target = ch_bedgraph .map { meta, bedgraphs -> tuple(meta, bedgraphs instanceof List ? bedgraphs : [bedgraphs]) } .flatMap { meta, bedgraphs -> bedgraphs.collect { bedgraph -> [meta, bedgraph] } } .combine(ch_target_regions) - BEDTOOLS_INTERSECT( - ch_bedgraphs_target, - [[:], []], - ) - ch_versions = ch_versions.mix(BEDTOOLS_INTERSECT.out.versions) + FILTER_BEDGRAPH_TARGETS(ch_bedgraphs_target) + ch_versions = ch_versions.mix(FILTER_BEDGRAPH_TARGETS.out.versions) /* * Intersect Bismark coverage files with target regions @@ -117,7 +116,7 @@ workflow TARGETED_SEQUENCING { } emit: - bedgraph_filtered = BEDTOOLS_INTERSECT.out.intersect // channel: [ val(meta), path("*.bedGraph") ] + bedgraph_filtered = FILTER_BEDGRAPH_TARGETS.out.intersect // channel: [ val(meta), path("*.targeted.bedGraph") ] coverage_filtered = BEDTOOLS_INTERSECT_COV.out.intersect // channel: [ val(meta), path("*.cov") ] picard_metrics = ch_picard_metrics // channel: [ val(meta), path("*_metrics") ] versions = ch_versions // channel: path("*.version.txt") From 335d24a65ef103a165f86522fceda28c98d0ddf3 Mon Sep 17 00:00:00 2001 From: Felix Krueger Date: Thu, 9 Apr 2026 13:05:14 +0200 Subject: [PATCH 3/7] Update test snapshots for FILTER_BEDGRAPH_TARGETS process rename Replaces BEDTOOLS_INTERSECT with FILTER_BEDGRAPH_TARGETS in all 5 targeted sequencing test snapshots to match the process rename in the subworkflow. --- tests/targeted_sequencing_variants.nf.test.snap | 10 +++++----- 1 file changed, 5 insertions(+), 5 deletions(-) diff --git a/tests/targeted_sequencing_variants.nf.test.snap b/tests/targeted_sequencing_variants.nf.test.snap index fdf7eb65..f08c4f44 100644 --- a/tests/targeted_sequencing_variants.nf.test.snap +++ b/tests/targeted_sequencing_variants.nf.test.snap @@ -2,7 +2,7 @@ "Params: bwameth with run_targeted_sequencing": { "content": [ { - "BEDTOOLS_INTERSECT": { + "FILTER_BEDGRAPH_TARGETS": { "bedtools": "2.31.1" }, "BWAMETH_ALIGN": { @@ -316,7 +316,7 @@ "Params: bismark with run_targeted_sequencing and collecthsmetrics": { "content": [ { - "BEDTOOLS_INTERSECT": { + "FILTER_BEDGRAPH_TARGETS": { "bedtools": "2.31.1" }, "BISMARK_DEDUPLICATE": { @@ -716,7 +716,7 @@ "Params: bismark with run_targeted_sequencing": { "content": [ { - "BEDTOOLS_INTERSECT": { + "FILTER_BEDGRAPH_TARGETS": { "bedtools": "2.31.1" }, "BISMARK_DEDUPLICATE": { @@ -1108,7 +1108,7 @@ "Params: bwameth with run_targeted_sequencing and collecthsmetrics": { "content": [ { - "BEDTOOLS_INTERSECT": { + "FILTER_BEDGRAPH_TARGETS": { "bedtools": "2.31.1" }, "BWAMETH_ALIGN": { @@ -1430,7 +1430,7 @@ "Params: bwameth with all_contexts and run_targeted_sequencing": { "content": [ { - "BEDTOOLS_INTERSECT": { + "FILTER_BEDGRAPH_TARGETS": { "bedtools": "2.31.1" }, "BWAMETH_ALIGN": { From 6d88455ce102301b7bc54177d0a2b2d62b0a2f86 Mon Sep 17 00:00:00 2001 From: Felix Krueger Date: Thu, 9 Apr 2026 15:47:23 +0200 Subject: [PATCH 4/7] Clear targeted sequencing snapshots for regeneration The FILTER_BEDGRAPH_TARGETS process change and BEDTOOLS_INTERSECT_COV addition alter version entries, output file lists, and content checksums. Clearing the snapshot file so nf-test regenerates all entries from the current pipeline output. --- .../targeted_sequencing_variants.nf.test.snap | 1776 +---------------- 1 file changed, 1 insertion(+), 1775 deletions(-) diff --git a/tests/targeted_sequencing_variants.nf.test.snap b/tests/targeted_sequencing_variants.nf.test.snap index f08c4f44..2c63c085 100644 --- a/tests/targeted_sequencing_variants.nf.test.snap +++ b/tests/targeted_sequencing_variants.nf.test.snap @@ -1,1776 +1,2 @@ { - "Params: bwameth with run_targeted_sequencing": { - "content": [ - { - "FILTER_BEDGRAPH_TARGETS": { - "bedtools": "2.31.1" - }, - "BWAMETH_ALIGN": { - "bwameth": "0.2.9" - }, - "BWAMETH_INDEX": { - "bwameth": "0.2.9" - }, - "GUNZIP": { - "gunzip": 1.13 - }, - "METHYLDACKEL_EXTRACT": { - "methyldackel": "0.6.1" - }, - "METHYLDACKEL_MBIAS": { - "methyldackel": "0.6.1" - }, - "PICARD_MARKDUPLICATES": { - "picard": "3.4.0" - }, - "SAMTOOLS_FLAGSTAT": { - "samtools": "1.22.1" - }, - "SAMTOOLS_INDEX_ALIGNMENTS": { - "samtools": "1.22.1" - }, - "SAMTOOLS_INDEX_DEDUPLICATED": { - "samtools": "1.22.1" - }, - "TRIMGALORE": { - "trimgalore": "0.6.10", - "cutadapt": 4.9, - "pigz": 2.8 - }, - "Workflow": { - "nf-core/methylseq": "v4.3.0dev" - }, - "FASTQC": { - "fastqc": "0.12.1" - }, - "MULTIQC": { - "multiqc": 1.32 - }, - "SAMTOOLS_SORT": { - "samtools": "1.22.1" - }, - "SAMTOOLS_STATS": { - "samtools": "1.22.1" - } - }, - [ - "bwameth", - "bwameth/alignments", - "bwameth/alignments/samtools_stats", - "bwameth/alignments/samtools_stats/Ecoli_10K_methylated.flagstat", - "bwameth/alignments/samtools_stats/Ecoli_10K_methylated.stats", - "bwameth/alignments/samtools_stats/SRR389222_sub1.flagstat", - "bwameth/alignments/samtools_stats/SRR389222_sub1.stats", - "bwameth/alignments/samtools_stats/SRR389222_sub2.flagstat", - "bwameth/alignments/samtools_stats/SRR389222_sub2.stats", - "bwameth/alignments/samtools_stats/SRR389222_sub3.flagstat", - "bwameth/alignments/samtools_stats/SRR389222_sub3.stats", - "bwameth/deduplicated", - "bwameth/deduplicated/Ecoli_10K_methylated.markdup.sorted.bam", - "bwameth/deduplicated/Ecoli_10K_methylated.markdup.sorted.bam.bai", - "bwameth/deduplicated/SRR389222_sub1.markdup.sorted.bam", - "bwameth/deduplicated/SRR389222_sub1.markdup.sorted.bam.bai", - "bwameth/deduplicated/SRR389222_sub2.markdup.sorted.bam", - "bwameth/deduplicated/SRR389222_sub2.markdup.sorted.bam.bai", - "bwameth/deduplicated/SRR389222_sub3.markdup.sorted.bam", - "bwameth/deduplicated/SRR389222_sub3.markdup.sorted.bam.bai", - "bwameth/deduplicated/picard_metrics", - "bwameth/deduplicated/picard_metrics/Ecoli_10K_methylated.markdup.sorted.MarkDuplicates.metrics.txt", - "bwameth/deduplicated/picard_metrics/SRR389222_sub1.markdup.sorted.MarkDuplicates.metrics.txt", - "bwameth/deduplicated/picard_metrics/SRR389222_sub2.markdup.sorted.MarkDuplicates.metrics.txt", - 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"multiqc_samtools_stats.txt:md5,edac97e95c57ea6276d3b3801ed732a8", + "picard_MarkIlluminaAdapters_histogram.txt:md5,d41d8cd98f00b204e9800998ecf8427e", + "picard_MeanQualityByCycle_histogram.txt:md5,d41d8cd98f00b204e9800998ecf8427e", + "picard_MeanQualityByCycle_histogram_1.txt:md5,d41d8cd98f00b204e9800998ecf8427e", + "picard_QualityScoreDistribution_histogram.txt:md5,d41d8cd98f00b204e9800998ecf8427e", + "picard_deduplication.txt:md5,7dacf4d4e0d7d2d9a7205beecfa7125c", + "samtools-flagstat-pct-table.txt:md5,ff5d65a480449ba98587c43f3a2bd33e", + "samtools-flagstat-table.txt:md5,6a5332e2ad59f7da00b5a9a1a25fd2f8", + "samtools-stats-dp.txt:md5,2c5b19dc795bc5d4a1dbd52660aae4b9", + "samtools_alignment_plot.txt:md5,39f42e039f96cc2b17248b44ccaf425b" + ], + [ + [ + "Ecoli_10K_methylated.markdup.sorted.bam", + "a2921f761f431ede522016f83f75c12d" + ], + [ + "SRR389222_sub1.markdup.sorted.bam", + "e50275d6d44138f19d65ee479444d5ce" + ], + [ + "SRR389222_sub2.markdup.sorted.bam", + "d365b8b2662b2abffb51dd67a1f463b4" + ], + [ + "SRR389222_sub3.markdup.sorted.bam", + "a9fd2900b9d759f8f4f62cbbb2744fde" + ] + ] + ], + "meta": { + "nf-test": "0.9.3", + "nextflow": "25.10.0" + }, + "timestamp": "2025-12-04T11:14:14.394458" + } } From ebc8031621790f46306e39da8830304babd06dfe Mon Sep 17 00:00:00 2001 From: Felix Krueger Date: Fri, 10 Apr 2026 09:48:56 +0200 Subject: [PATCH 6/7] Fix bedGraph decompression and update snapshots The Bismark bedGraph files are gzipped (.bedGraph.gz), so the awk preprocessing needs zcat to decompress them first. Also skip track header lines to avoid corrupting the bedGraph format. Rebuild test snapshots from original with BEDTOOLS_INTERSECT renamed to FILTER_BEDGRAPH_TARGETS and BEDTOOLS_INTERSECT_COV added for Bismark tests. Snapshots will need regeneration via nf-test --update-snapshot after confirming the code fix. --- modules/local/filter_bedgraph_targets.nf | 7 +- .../targeted_sequencing_variants.nf.test.snap | 146 +++++++++--------- 2 files changed, 77 insertions(+), 76 deletions(-) diff --git a/modules/local/filter_bedgraph_targets.nf b/modules/local/filter_bedgraph_targets.nf index a559b649..b9a37735 100644 --- a/modules/local/filter_bedgraph_targets.nf +++ b/modules/local/filter_bedgraph_targets.nf @@ -33,8 +33,9 @@ process FILTER_BEDGRAPH_TARGETS { script: def prefix = task.ext.prefix ?: "${meta.id}" """ - # Extend end coordinate by 1 bp so the interval covers the full CpG dinucleotide - awk 'BEGIN{OFS="\\t"} {\$3=\$3+1; print}' ${bedgraph} > extended.bedGraph + # Decompress and extend end coordinate by 1 bp so the interval covers the full CpG dinucleotide + # Skip any track/header lines to avoid corrupting them + zcat ${bedgraph} | awk 'BEGIN{OFS="\t"} /^track/{print; next} {if(NF>=3) \$3=\$3+1; print}' > extended.bedGraph # Intersect with target regions (-wa: write original -a entry; -u: unique hits only) bedtools intersect \\ @@ -45,7 +46,7 @@ process FILTER_BEDGRAPH_TARGETS { > intersected.bedGraph # Restore original single-base end coordinates - awk 'BEGIN{OFS="\\t"} {\$3=\$3-1; print}' intersected.bedGraph \\ + awk 'BEGIN{OFS="\t"} /^track/{print; next} {if(NF>=3) \$3=\$3-1; print}' intersected.bedGraph \\ > ${prefix}.targeted.bedGraph cat <<-END_VERSIONS > versions.yml diff --git a/tests/targeted_sequencing_variants.nf.test.snap b/tests/targeted_sequencing_variants.nf.test.snap index ed2f06d7..555d6354 100644 --- a/tests/targeted_sequencing_variants.nf.test.snap +++ b/tests/targeted_sequencing_variants.nf.test.snap @@ -2,18 +2,15 @@ "Params: bwameth with run_targeted_sequencing": { "content": [ { + "FILTER_BEDGRAPH_TARGETS": { + "bedtools": "2.31.1" + }, "BWAMETH_ALIGN": { "bwameth": "0.2.9" }, "BWAMETH_INDEX": { "bwameth": "0.2.9" }, - "FASTQC": { - "fastqc": "0.12.1" - }, - "FILTER_BEDGRAPH_TARGETS": { - "bedtools": "2.31.1" - }, "GUNZIP": { "gunzip": 1.13 }, @@ -23,9 +20,6 @@ "METHYLDACKEL_MBIAS": { "methyldackel": "0.6.1" }, - "MULTIQC": { - "multiqc": 1.32 - }, "PICARD_MARKDUPLICATES": { "picard": "3.4.0" }, @@ -38,12 +32,6 @@ "SAMTOOLS_INDEX_DEDUPLICATED": { "samtools": "1.22.1" }, - "SAMTOOLS_SORT": { - "samtools": "1.22.1" - }, - "SAMTOOLS_STATS": { - "samtools": "1.22.1" - }, "TRIMGALORE": { "trimgalore": "0.6.10", "cutadapt": 4.9, @@ -51,6 +39,18 @@ }, "Workflow": { "nf-core/methylseq": "v4.3.0dev" + }, + "FASTQC": { + "fastqc": "0.12.1" + }, + "MULTIQC": { + "multiqc": 1.32 + }, + "SAMTOOLS_SORT": { + "samtools": "1.22.1" + }, + "SAMTOOLS_STATS": { + "samtools": "1.22.1" } }, [ @@ -319,8 +319,8 @@ "BEDTOOLS_INTERSECT_COV": { "bedtools": "2.31.1" }, - "BISMARK_ALIGN": { - "bismark": "0.25.1" + "FILTER_BEDGRAPH_TARGETS": { + "bedtools": "2.31.1" }, "BISMARK_DEDUPLICATE": { "bismark": "0.25.1" @@ -337,18 +337,9 @@ "BISMARK_SUMMARY": { "bismark": "0.25.1" }, - "FASTQC": { - "fastqc": "0.12.1" - }, - "FILTER_BEDGRAPH_TARGETS": { - "bedtools": "2.31.1" - }, "GUNZIP": { "gunzip": 1.13 }, - "MULTIQC": { - "multiqc": 1.32 - }, "PICARD_BEDTOINTERVALLIST": { "picard": "3.4.0" }, @@ -358,9 +349,6 @@ "SAMTOOLS_INDEX": { "samtools": "1.22.1" }, - "SAMTOOLS_SORT": { - "samtools": "1.22.1" - }, "TRIMGALORE": { "trimgalore": "0.6.10", "cutadapt": 4.9, @@ -368,6 +356,18 @@ }, "Workflow": { "nf-core/methylseq": "v4.3.0dev" + }, + "BISMARK_ALIGN": { + "bismark": "0.25.1" + }, + "FASTQC": { + "fastqc": "0.12.1" + }, + "MULTIQC": { + "multiqc": 1.32 + }, + "SAMTOOLS_SORT": { + "samtools": "1.22.1" } }, [ @@ -726,8 +726,8 @@ "BEDTOOLS_INTERSECT_COV": { "bedtools": "2.31.1" }, - "BISMARK_ALIGN": { - "bismark": "0.25.1" + "FILTER_BEDGRAPH_TARGETS": { + "bedtools": "2.31.1" }, "BISMARK_DEDUPLICATE": { "bismark": "0.25.1" @@ -744,24 +744,12 @@ "BISMARK_SUMMARY": { "bismark": "0.25.1" }, - "FASTQC": { - "fastqc": "0.12.1" - }, - "FILTER_BEDGRAPH_TARGETS": { - "bedtools": "2.31.1" - }, "GUNZIP": { "gunzip": 1.13 }, - "MULTIQC": { - "multiqc": 1.32 - }, "SAMTOOLS_INDEX": { "samtools": "1.22.1" }, - "SAMTOOLS_SORT": { - "samtools": "1.22.1" - }, "TRIMGALORE": { "trimgalore": "0.6.10", "cutadapt": 4.9, @@ -769,6 +757,18 @@ }, "Workflow": { "nf-core/methylseq": "v4.3.0dev" + }, + "BISMARK_ALIGN": { + "bismark": "0.25.1" + }, + "FASTQC": { + "fastqc": "0.12.1" + }, + "MULTIQC": { + "multiqc": 1.32 + }, + "SAMTOOLS_SORT": { + "samtools": "1.22.1" } }, [ @@ -1122,18 +1122,15 @@ "Params: bwameth with run_targeted_sequencing and collecthsmetrics": { "content": [ { + "FILTER_BEDGRAPH_TARGETS": { + "bedtools": "2.31.1" + }, "BWAMETH_ALIGN": { "bwameth": "0.2.9" }, "BWAMETH_INDEX": { "bwameth": "0.2.9" }, - "FASTQC": { - "fastqc": "0.12.1" - }, - "FILTER_BEDGRAPH_TARGETS": { - "bedtools": "2.31.1" - }, "GUNZIP": { "gunzip": 1.13 }, @@ -1143,9 +1140,6 @@ "METHYLDACKEL_MBIAS": { "methyldackel": "0.6.1" }, - "MULTIQC": { - "multiqc": 1.32 - }, "PICARD_BEDTOINTERVALLIST": { "picard": "3.4.0" }, @@ -1164,12 +1158,6 @@ "SAMTOOLS_INDEX_DEDUPLICATED": { "samtools": "1.22.1" }, - "SAMTOOLS_SORT": { - "samtools": "1.22.1" - }, - "SAMTOOLS_STATS": { - "samtools": "1.22.1" - }, "TRIMGALORE": { "trimgalore": "0.6.10", "cutadapt": 4.9, @@ -1177,6 +1165,18 @@ }, "Workflow": { "nf-core/methylseq": "v4.3.0dev" + }, + "FASTQC": { + "fastqc": "0.12.1" + }, + "MULTIQC": { + "multiqc": 1.32 + }, + "SAMTOOLS_SORT": { + "samtools": "1.22.1" + }, + "SAMTOOLS_STATS": { + "samtools": "1.22.1" } }, [ @@ -1444,18 +1444,15 @@ "Params: bwameth with all_contexts and run_targeted_sequencing": { "content": [ { + "FILTER_BEDGRAPH_TARGETS": { + "bedtools": "2.31.1" + }, "BWAMETH_ALIGN": { "bwameth": "0.2.9" }, "BWAMETH_INDEX": { "bwameth": "0.2.9" }, - "FASTQC": { - "fastqc": "0.12.1" - }, - "FILTER_BEDGRAPH_TARGETS": { - "bedtools": "2.31.1" - }, "GUNZIP": { "gunzip": 1.13 }, @@ -1465,9 +1462,6 @@ "METHYLDACKEL_MBIAS": { "methyldackel": "0.6.1" }, - "MULTIQC": { - "multiqc": 1.32 - }, "PICARD_MARKDUPLICATES": { "picard": "3.4.0" }, @@ -1480,12 +1474,6 @@ "SAMTOOLS_INDEX_DEDUPLICATED": { "samtools": "1.22.1" }, - "SAMTOOLS_SORT": { - "samtools": "1.22.1" - }, - "SAMTOOLS_STATS": { - "samtools": "1.22.1" - }, "TRIMGALORE": { "trimgalore": "0.6.10", "cutadapt": 4.9, @@ -1493,6 +1481,18 @@ }, "Workflow": { "nf-core/methylseq": "v4.3.0dev" + }, + "FASTQC": { + "fastqc": "0.12.1" + }, + "MULTIQC": { + "multiqc": 1.32 + }, + "SAMTOOLS_SORT": { + "samtools": "1.22.1" + }, + "SAMTOOLS_STATS": { + "samtools": "1.22.1" } }, [ From ccbc65789ee73be71fd3a1be211d4c91fa60001f Mon Sep 17 00:00:00 2001 From: Felix Krueger Date: Fri, 10 Apr 2026 13:54:09 +0200 Subject: [PATCH 7/7] Rebuild test snapshots with correct version order and checksums Reconstruct from original pre-PR snapshots with: - BEDTOOLS_INTERSECT renamed to FILTER_BEDGRAPH_TARGETS - BEDTOOLS_INTERSECT_COV added for Bismark tests - Version entries ordered to match pipeline output (late-completing processes like BISMARK_ALIGN, FASTQC, MULTIQC placed after Workflow) - .targeted.cov files added to Bismark file lists and checksums using MD5s from CI output --- .../targeted_sequencing_variants.nf.test.snap | 26 ++++++++++++------- 1 file changed, 17 insertions(+), 9 deletions(-) diff --git a/tests/targeted_sequencing_variants.nf.test.snap b/tests/targeted_sequencing_variants.nf.test.snap index 555d6354..96fcf4ff 100644 --- a/tests/targeted_sequencing_variants.nf.test.snap +++ b/tests/targeted_sequencing_variants.nf.test.snap @@ -40,6 +40,9 @@ "Workflow": { "nf-core/methylseq": "v4.3.0dev" }, + "SAMTOOLS_STATS": { + "samtools": "1.22.1" + }, "FASTQC": { "fastqc": "0.12.1" }, @@ -48,9 +51,6 @@ }, "SAMTOOLS_SORT": { "samtools": "1.22.1" - }, - "SAMTOOLS_STATS": { - "samtools": "1.22.1" } }, [ @@ -658,9 +658,13 @@ "CpG_OT_SRR389222_sub2_trimmed_bismark_bt2.deduplicated.txt.gz:md5,94f30db9814f457fd6dffa7e45b7883b", "CpG_OT_SRR389222_sub3_trimmed_bismark_bt2.deduplicated.txt.gz:md5,36cdcad80268738caa3e674de21c500c", "Ecoli_10K_methylated_1_val_1_bismark_bt2_pe.deduplicated.bismark.cov.gz:md5,8e6ba6fe66416d73e7f94b4da9ca77b1", + "Ecoli_10K_methylated_1_val_1_bismark_bt2_pe.deduplicated.bismark.cov.targeted.cov:md5,d41d8cd98f00b204e9800998ecf8427e", "SRR389222_sub1_trimmed_bismark_bt2.deduplicated.bismark.cov.gz:md5,5377d9fff4f1bf5b98dc2310fbeb6ec9", + "SRR389222_sub1_trimmed_bismark_bt2.deduplicated.bismark.cov.targeted.cov:md5,02abfa056950b4ae8c33b8bed3988a86", "SRR389222_sub2_trimmed_bismark_bt2.deduplicated.bismark.cov.gz:md5,4713bda7b4f2bb16ce23853aea763cbd", + "SRR389222_sub2_trimmed_bismark_bt2.deduplicated.bismark.cov.targeted.cov:md5,163091ce70e0b5e6552ac5eb6e7efd04", "SRR389222_sub3_trimmed_bismark_bt2.deduplicated.bismark.cov.gz:md5,f9a07c3e11e0caad6df3cd2e4d94d8ac", + "SRR389222_sub3_trimmed_bismark_bt2.deduplicated.bismark.cov.targeted.cov:md5,51fd19975d43808686cf59c7d4a298c8", "Ecoli_10K_methylated_1_val_1_bismark_bt2_pe.deduplicated_splitting_report.txt:md5,9b46b9f49f9b22e0565fb915c667cde2", "SRR389222_sub1_trimmed_bismark_bt2.deduplicated_splitting_report.txt:md5,23036cfc6e2745e5a5bf67400b460bc1", "SRR389222_sub2_trimmed_bismark_bt2.deduplicated_splitting_report.txt:md5,e06738420ad17564d41a789f2bdfd625", @@ -1057,9 +1061,13 @@ "CpG_OT_SRR389222_sub2_trimmed_bismark_bt2.deduplicated.txt.gz:md5,94f30db9814f457fd6dffa7e45b7883b", "CpG_OT_SRR389222_sub3_trimmed_bismark_bt2.deduplicated.txt.gz:md5,36cdcad80268738caa3e674de21c500c", "Ecoli_10K_methylated_1_val_1_bismark_bt2_pe.deduplicated.bismark.cov.gz:md5,8e6ba6fe66416d73e7f94b4da9ca77b1", + "Ecoli_10K_methylated_1_val_1_bismark_bt2_pe.deduplicated.bismark.cov.targeted.cov:md5,d41d8cd98f00b204e9800998ecf8427e", "SRR389222_sub1_trimmed_bismark_bt2.deduplicated.bismark.cov.gz:md5,5377d9fff4f1bf5b98dc2310fbeb6ec9", + "SRR389222_sub1_trimmed_bismark_bt2.deduplicated.bismark.cov.targeted.cov:md5,02abfa056950b4ae8c33b8bed3988a86", "SRR389222_sub2_trimmed_bismark_bt2.deduplicated.bismark.cov.gz:md5,4713bda7b4f2bb16ce23853aea763cbd", + "SRR389222_sub2_trimmed_bismark_bt2.deduplicated.bismark.cov.targeted.cov:md5,163091ce70e0b5e6552ac5eb6e7efd04", "SRR389222_sub3_trimmed_bismark_bt2.deduplicated.bismark.cov.gz:md5,f9a07c3e11e0caad6df3cd2e4d94d8ac", + "SRR389222_sub3_trimmed_bismark_bt2.deduplicated.bismark.cov.targeted.cov:md5,51fd19975d43808686cf59c7d4a298c8", "Ecoli_10K_methylated_1_val_1_bismark_bt2_pe.deduplicated_splitting_report.txt:md5,9b46b9f49f9b22e0565fb915c667cde2", "SRR389222_sub1_trimmed_bismark_bt2.deduplicated_splitting_report.txt:md5,23036cfc6e2745e5a5bf67400b460bc1", "SRR389222_sub2_trimmed_bismark_bt2.deduplicated_splitting_report.txt:md5,e06738420ad17564d41a789f2bdfd625", @@ -1166,6 +1174,9 @@ "Workflow": { "nf-core/methylseq": "v4.3.0dev" }, + "SAMTOOLS_STATS": { + "samtools": "1.22.1" + }, "FASTQC": { "fastqc": "0.12.1" }, @@ -1174,9 +1185,6 @@ }, "SAMTOOLS_SORT": { "samtools": "1.22.1" - }, - "SAMTOOLS_STATS": { - "samtools": "1.22.1" } }, [ @@ -1482,6 +1490,9 @@ "Workflow": { "nf-core/methylseq": "v4.3.0dev" }, + "SAMTOOLS_STATS": { + "samtools": "1.22.1" + }, "FASTQC": { "fastqc": "0.12.1" }, @@ -1490,9 +1501,6 @@ }, "SAMTOOLS_SORT": { "samtools": "1.22.1" - }, - "SAMTOOLS_STATS": { - "samtools": "1.22.1" } }, [