Filter Bismark coverage files in targeted sequencing mode#604
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FelixKrueger merged 7 commits intonf-core:devfrom Apr 10, 2026
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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.
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Warning Newer version of the nf-core template is available. Your pipeline is using an old version of the nf-core template: 3.5.1. For more documentation on how to update your pipeline, please see the nf-core documentation and Synchronisation documentation. |
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.
Replaces BEDTOOLS_INTERSECT with FILTER_BEDGRAPH_TARGETS in all 5 targeted sequencing test snapshots to match the process rename in the subworkflow.
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.
- Rename BEDTOOLS_INTERSECT to FILTER_BEDGRAPH_TARGETS in version entries - Add BEDTOOLS_INTERSECT_COV version entry for Bismark tests - Add .targeted.cov output files to Bismark test file lists - Sort version keys alphabetically to match pipeline output order
ryanckelly44
reviewed
Apr 9, 2026
| 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 |
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Produces an empty bedGraph file if gzipped file is passed to awk.
Change to:
zcat ${bedgraph} | awk 'BEGIN{OFS="\t"} {$3=$3+1; print}' \ > extended.bedGraph
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.
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
maxulysse
approved these changes
Apr 10, 2026
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Summary
--run_targeted_sequencingis enabled, only bedGraph files were filtered against the target BED. The.cov.gzfiles from Bismark were published unfiltered, containing off-target CpGs.BEDTOOLS_INTERSECT_COVprocess alias to filter.covfiles identically to bedGraphs, so downstream tools (methylKit, bsseq, DSS) receive only on-target sites..covfiles).Changes
subworkflows/local/targeted_sequencing/main.nf: AddedBEDTOOLS_INTERSECT_COValias, newch_coverageinput, intersect call, andcoverage_filteredemitworkflows/methylseq/main.nf: Passmethylation_coveragefrom Bismark subworkflow toTARGETED_SEQUENCINGconf/modules/bedtools_intersect_cov.config: Config for aliased process (ext.suffix = 'targeted.cov', publishDir tomethylation_coverage/)conf/subworkflows/targeted_sequencing.config: Include new configTest plan
--aligner bismark --run_targeted_sequencing --target_regions_file <bed>and verify*.targeted.covfiles appear inbismark/methylation_calls/methylation_coverage/.covfall within target regions (bedtools intersect -vshould return 0 lines)--aligner bwameth --run_targeted_sequencingand verify no errors (empty coverage channel is handled gracefully)