diff --git a/modules/nf-core/rastair2/call/main.nf b/modules/nf-core/rastair2/call/main.nf
index a4feaa923128..3986506a80c3 100644
--- a/modules/nf-core/rastair2/call/main.nf
+++ b/modules/nf-core/rastair2/call/main.nf
@@ -8,7 +8,7 @@ process RASTAIR2_CALL {
: 'community.wave.seqera.io/library/htslib_rastair:4ffd26b40813e268'}"
input:
- tuple val(meta), path(bam), path(index)
+ tuple val(meta), path(bam), path(index), path(mbias_cutoffs)
tuple val(meta2), path(fasta), path(fai)
output:
@@ -27,10 +27,17 @@ process RASTAIR2_CALL {
def prefix = task.ext.prefix ?: "${meta.id}"
// Single-end reads carry no pairing flags: strand comes from the alignment direction alone.
def unpaired = meta.single_end ? '--unpaired' : ''
+ // Bases to ignore at read ends, as suggested by rastair2/mbias: line 1 is --nOT, line 2 --nOB
+ def cutoffs_files = mbias_cutoffs instanceof List ? mbias_cutoffs : [mbias_cutoffs]
+ if (cutoffs_files.size() > 1) {
+ error("rastair2/call takes one M-bias cutoffs file per sample, got ${cutoffs_files.size()}: restrict rastair2/mbias to one contig with --region")
+ }
+ def trim = cutoffs_files ? "--nOT \$(sed -n 1p ${mbias_cutoffs}) --nOB \$(sed -n 2p ${mbias_cutoffs})" : ''
"""
rastair call \\
${args} \\
${unpaired} \\
+ ${trim} \\
--threads ${task.cpus} \\
--fasta-file ${fasta} \\
--vcf ${prefix}.vcf.gz \\
diff --git a/modules/nf-core/rastair2/call/meta.yml b/modules/nf-core/rastair2/call/meta.yml
index 31c8905af76a..c7f73f55ec79 100644
--- a/modules/nf-core/rastair2/call/meta.yml
+++ b/modules/nf-core/rastair2/call/meta.yml
@@ -51,6 +51,15 @@ input:
pattern: "*.{bai,csi,crai}"
ontologies:
- edam: "http://edamontology.org/format_3327"
+ - mbias_cutoffs:
+ type: file
+ description: |
+ Optional. One M-bias cutoffs file from rastair2/mbias (pass `[]` for none).
+ Line 1 is used as `--nOT` and line 2 as `--nOB`: bases to ignore at the
+ read ends, as `r1_start,r1_end,r2_start,r2_end`.
+ pattern: "*.cutoffs.txt"
+ ontologies:
+ - edam: "http://edamontology.org/format_3475"
- - meta2:
type: map
description: |
diff --git a/modules/nf-core/rastair2/call/tests/main.nf.test b/modules/nf-core/rastair2/call/tests/main.nf.test
index 5530559ad49c..3a5015e63180 100644
--- a/modules/nf-core/rastair2/call/tests/main.nf.test
+++ b/modules/nf-core/rastair2/call/tests/main.nf.test
@@ -8,6 +8,7 @@ nextflow_process {
tag "modules_nfcore"
tag "rastair2"
tag "rastair2/call"
+ tag "rastair2/mbias"
// NOTE The VCF header records the command line (including --threads), so only the #CHROM line
// and records are snapshotted. They are hashed as text because rastair declares Number=M
@@ -18,12 +19,13 @@ nextflow_process {
when {
process {
"""
- input[0] = Channel.of([
+ input[0] = channel.of([
[ id:'test', single_end:false ],
file(params.modules_testdata_base_path + 'genomics/sarscov2/illumina/bam/test.paired_end.methylated.sorted.bam', checkIfExists: true),
- file(params.modules_testdata_base_path + 'genomics/sarscov2/illumina/bam/test.paired_end.methylated.sorted.bam.bai', checkIfExists: true)
+ file(params.modules_testdata_base_path + 'genomics/sarscov2/illumina/bam/test.paired_end.methylated.sorted.bam.bai', checkIfExists: true),
+ []
])
- input[1] = Channel.of([
+ input[1] = channel.of([
[ id:'genome' ],
file(params.modules_testdata_base_path + 'genomics/sarscov2/genome/genome.fasta', checkIfExists: true),
file(params.modules_testdata_base_path + 'genomics/sarscov2/genome/genome.fasta.fai', checkIfExists: true)
@@ -33,11 +35,11 @@ nextflow_process {
}
then {
+ assert process.success
assertAll(
- { assert process.success },
- { assert path(process.out.vcf[0][1]).linesGzip.any { it.startsWith('##rastairVersion=') } },
+ { assert path(process.out.vcf[0][1]).linesGzip.any { line -> line.startsWith('##rastairVersion=') } },
{ assert snapshot(
- path(process.out.vcf[0][1]).linesGzip.findAll { !it.startsWith('##') }.join('\n').md5(),
+ path(process.out.vcf[0][1]).linesGzip.findAll { line -> !line.startsWith('##') }.join('\n').md5(),
process.out.tbi.collect { meta, tbi -> file(tbi).name },
process.out.bed,
process.out.bed_tbi.collect { meta, tbi -> file(tbi).name },
@@ -52,12 +54,13 @@ nextflow_process {
when {
process {
"""
- input[0] = Channel.of([
+ input[0] = channel.of([
[ id:'test', single_end:true ],
file(params.modules_testdata_base_path + 'genomics/sarscov2/illumina/bam/test.single_end.sorted.bam', checkIfExists: true),
- file(params.modules_testdata_base_path + 'genomics/sarscov2/illumina/bam/test.single_end.sorted.bam.bai', checkIfExists: true)
+ file(params.modules_testdata_base_path + 'genomics/sarscov2/illumina/bam/test.single_end.sorted.bam.bai', checkIfExists: true),
+ []
])
- input[1] = Channel.of([
+ input[1] = channel.of([
[ id:'genome' ],
file(params.modules_testdata_base_path + 'genomics/sarscov2/genome/genome.fasta', checkIfExists: true),
file(params.modules_testdata_base_path + 'genomics/sarscov2/genome/genome.fasta.fai', checkIfExists: true)
@@ -67,11 +70,11 @@ nextflow_process {
}
then {
+ assert process.success
assertAll(
- { assert process.success },
- { assert path(process.out.vcf[0][1]).linesGzip.any { it.startsWith('##rastairCommand=') && it.contains('--unpaired') } },
+ { assert path(process.out.vcf[0][1]).linesGzip.any { line -> line.startsWith('##rastairCommand=') && line.contains('--unpaired') } },
{ assert snapshot(
- path(process.out.vcf[0][1]).linesGzip.findAll { !it.startsWith('##') }.join('\n').md5(),
+ path(process.out.vcf[0][1]).linesGzip.findAll { line -> !line.startsWith('##') }.join('\n').md5(),
process.out.tbi.collect { meta, tbi -> file(tbi).name },
process.out.bed,
process.out.bed_tbi.collect { meta, tbi -> file(tbi).name },
@@ -86,12 +89,13 @@ nextflow_process {
when {
process {
"""
- input[0] = Channel.of([
+ input[0] = channel.of([
[ id:'test', single_end:false ],
file(params.modules_testdata_base_path + 'genomics/homo_sapiens/illumina/cram/test.paired_end.sorted.cram', checkIfExists: true),
- file(params.modules_testdata_base_path + 'genomics/homo_sapiens/illumina/cram/test.paired_end.sorted.cram.crai', checkIfExists: true)
+ file(params.modules_testdata_base_path + 'genomics/homo_sapiens/illumina/cram/test.paired_end.sorted.cram.crai', checkIfExists: true),
+ []
])
- input[1] = Channel.of([
+ input[1] = channel.of([
[ id:'genome' ],
file(params.modules_testdata_base_path + 'genomics/homo_sapiens/genome/genome.fasta', checkIfExists: true),
file(params.modules_testdata_base_path + 'genomics/homo_sapiens/genome/genome.fasta.fai', checkIfExists: true)
@@ -101,11 +105,65 @@ nextflow_process {
}
then {
+ assert process.success
assertAll(
- { assert process.success },
- { assert path(process.out.vcf[0][1]).linesGzip.any { !it.startsWith('#') } },
+ { assert path(process.out.vcf[0][1]).linesGzip.any { line -> !line.startsWith('#') } },
{ assert snapshot(
- path(process.out.vcf[0][1]).linesGzip.findAll { !it.startsWith('##') }.join('\n').md5(),
+ path(process.out.vcf[0][1]).linesGzip.findAll { line -> !line.startsWith('##') }.join('\n').md5(),
+ process.out.tbi.collect { meta, tbi -> file(tbi).name },
+ process.out.bed,
+ process.out.bed_tbi.collect { meta, tbi -> file(tbi).name },
+ process.out.findAll { key, val -> key.startsWith("versions") }
+ ).match() }
+ )
+ }
+ }
+
+ test("sarscov2 - paired-end methylated bam - mbias cutoffs") {
+
+ setup {
+ run("RASTAIR2_MBIAS") {
+ script "../../mbias/main.nf"
+ process {
+ """
+ input[0] = channel.of([
+ [ id:'test', single_end:false ],
+ file(params.modules_testdata_base_path + 'genomics/sarscov2/illumina/bam/test.paired_end.methylated.sorted.bam', checkIfExists: true),
+ file(params.modules_testdata_base_path + 'genomics/sarscov2/illumina/bam/test.paired_end.methylated.sorted.bam.bai', checkIfExists: true)
+ ])
+ input[1] = channel.of([
+ [ id:'genome' ],
+ file(params.modules_testdata_base_path + 'genomics/sarscov2/genome/genome.fasta', checkIfExists: true),
+ file(params.modules_testdata_base_path + 'genomics/sarscov2/genome/genome.fasta.fai', checkIfExists: true)
+ ])
+ """
+ }
+ }
+ }
+
+ when {
+ process {
+ """
+ input[0] = channel.of([
+ [ id:'test', single_end:false ],
+ file(params.modules_testdata_base_path + 'genomics/sarscov2/illumina/bam/test.paired_end.methylated.sorted.bam', checkIfExists: true),
+ file(params.modules_testdata_base_path + 'genomics/sarscov2/illumina/bam/test.paired_end.methylated.sorted.bam.bai', checkIfExists: true)
+ ]).join(RASTAIR2_MBIAS.out.cutoffs)
+ input[1] = channel.of([
+ [ id:'genome' ],
+ file(params.modules_testdata_base_path + 'genomics/sarscov2/genome/genome.fasta', checkIfExists: true),
+ file(params.modules_testdata_base_path + 'genomics/sarscov2/genome/genome.fasta.fai', checkIfExists: true)
+ ])
+ """
+ }
+ }
+
+ then {
+ assert process.success
+ assertAll(
+ { assert path(process.out.vcf[0][1]).linesGzip.any { line -> line.startsWith('##rastairCommand=') && line.contains('--nOT') && line.contains('--nOB') } },
+ { assert snapshot(
+ path(process.out.vcf[0][1]).linesGzip.findAll { line -> !line.startsWith('##') }.join('\n').md5(),
process.out.tbi.collect { meta, tbi -> file(tbi).name },
process.out.bed,
process.out.bed_tbi.collect { meta, tbi -> file(tbi).name },
@@ -122,12 +180,13 @@ nextflow_process {
when {
process {
"""
- input[0] = Channel.of([
+ input[0] = channel.of([
[ id:'test', single_end:false ],
file(params.modules_testdata_base_path + 'genomics/sarscov2/illumina/bam/test.paired_end.methylated.sorted.bam', checkIfExists: true),
- file(params.modules_testdata_base_path + 'genomics/sarscov2/illumina/bam/test.paired_end.methylated.sorted.bam.bai', checkIfExists: true)
+ file(params.modules_testdata_base_path + 'genomics/sarscov2/illumina/bam/test.paired_end.methylated.sorted.bam.bai', checkIfExists: true),
+ []
])
- input[1] = Channel.of([
+ input[1] = channel.of([
[ id:'genome' ],
file(params.modules_testdata_base_path + 'genomics/sarscov2/genome/genome.fasta', checkIfExists: true),
file(params.modules_testdata_base_path + 'genomics/sarscov2/genome/genome.fasta.fai', checkIfExists: true)
@@ -137,8 +196,8 @@ nextflow_process {
}
then {
+ assert process.success
assertAll(
- { assert process.success },
{ assert snapshot(sanitizeOutput(process.out)).match() }
)
}
diff --git a/modules/nf-core/rastair2/call/tests/main.nf.test.snap b/modules/nf-core/rastair2/call/tests/main.nf.test.snap
index d6d4eb9f736c..1e55a5f1039c 100644
--- a/modules/nf-core/rastair2/call/tests/main.nf.test.snap
+++ b/modules/nf-core/rastair2/call/tests/main.nf.test.snap
@@ -142,6 +142,47 @@
"nextflow": "26.10.5"
}
},
+ "sarscov2 - paired-end methylated bam - mbias cutoffs": {
+ "content": [
+ "d1630262a4ab6255e9e441e6857e746f",
+ [
+ "test.vcf.gz.tbi"
+ ],
+ [
+ [
+ {
+ "id": "test",
+ "single_end": false
+ },
+ "test.bed.gz:md5,ed8237adc7795a7485dab20c69146675"
+ ]
+ ],
+ [
+ "test.bed.gz.tbi"
+ ],
+ {
+ "versions_rastair": [
+ [
+ "RASTAIR2_CALL",
+ "rastair",
+ "2.2.0"
+ ]
+ ],
+ "versions_tabix": [
+ [
+ "RASTAIR2_CALL",
+ "tabix",
+ "1.24"
+ ]
+ ]
+ }
+ ],
+ "timestamp": "2026-11-10T10:58:57.116513",
+ "meta": {
+ "nf-test": "0.9.5",
+ "nextflow": "26.10.5"
+ }
+ },
"sarscov2 - single-end bam": {
"content": [
"1b3f2f0fde4de41a51853252c8ba063f",
diff --git a/modules/nf-core/rastair2/mbias/.conda-lock/linux_amd64-bd-4ffd26b40813e268_1.txt b/modules/nf-core/rastair2/mbias/.conda-lock/linux_amd64-bd-4ffd26b40813e268_1.txt
new file mode 100644
index 000000000000..6295da4535d5
--- /dev/null
+++ b/modules/nf-core/rastair2/mbias/.conda-lock/linux_amd64-bd-4ffd26b40813e268_1.txt
@@ -0,0 +1,2248 @@
+version: 6
+environments:
+ default:
+ channels:
+ - url: https://conda.anaconda.org/conda-forge/
+ - url: https://conda.anaconda.org/bioconda/
+ - url: https://conda.anaconda.org/bioconda/
+ options:
+ pypi-prerelease-mode: if-necessary-or-explicit
+ packages:
+ linux-64:
+ - conda: https://conda.anaconda.org/conda-forge/linux-64/_openmp_mutex-4.5-20_gnu.conda
+ - conda: https://conda.anaconda.org/conda-forge/noarch/_r-mutex-1.0.1-anacondar_1.tar.bz2
+ - conda: https://conda.anaconda.org/bioconda/linux-64/bcftools-1.24-h487d631_1.conda
+ - conda: https://conda.anaconda.org/conda-forge/linux-64/binutils_impl_linux-64-2.46.1-default_hfdba357_103.conda
+ - conda: https://conda.anaconda.org/conda-forge/linux-64/binutils_linux-64-2.46.1-default_h4852527_103.conda
+ - conda: https://conda.anaconda.org/bioconda/noarch/bioconductor-biocgenerics-0.58.1-r45hdfd78af_0.conda
+ - conda: https://conda.anaconda.org/bioconda/linux-64/bioconductor-biocparallel-1.46.0-r45h55fe1e3_0.conda
+ - conda: https://conda.anaconda.org/bioconda/linux-64/bioconductor-biostrings-2.80.2-r45h262fe30_0.conda
+ - conda: https://conda.anaconda.org/bioconda/linux-64/bioconductor-genomicranges-1.64.0-r45h262fe30_0.conda
+ - conda: https://conda.anaconda.org/bioconda/linux-64/bioconductor-iranges-2.46.0-r45h262fe30_0.conda
+ - conda: https://conda.anaconda.org/bioconda/linux-64/bioconductor-rhtslib-3.8.0-r45h262fe30_0.conda
+ - conda: https://conda.anaconda.org/bioconda/linux-64/bioconductor-rsamtools-2.28.0-r45h55fe1e3_0.conda
+ - conda: https://conda.anaconda.org/bioconda/linux-64/bioconductor-s4vectors-0.50.3-r45h262fe30_0.conda
+ - conda: https://conda.anaconda.org/bioconda/noarch/bioconductor-seqinfo-1.2.0-r45hdfd78af_0.conda
+ - conda: https://conda.anaconda.org/bioconda/linux-64/bioconductor-xvector-0.52.0-r45h262fe30_0.conda
+ - conda: https://conda.anaconda.org/conda-forge/linux-64/bwidget-1.10.1-hfc2019e_3.conda
+ - conda: https://conda.anaconda.org/conda-forge/linux-64/bzip2-1.0.8-hda65f42_10.conda
+ - conda: https://conda.anaconda.org/conda-forge/linux-64/c-ares-1.34.9-hebe6cf0_0.conda
+ - conda: https://conda.anaconda.org/conda-forge/noarch/ca-certificates-2026.10.23-hbd8a1cb_0.conda
+ - conda: https://conda.anaconda.org/conda-forge/linux-64/cairo-1.18.6-h3c89d7e_0.conda
+ - conda: https://conda.anaconda.org/conda-forge/linux-64/curl-8.23.0-ha042cf0_0.conda
+ - conda: https://conda.anaconda.org/conda-forge/noarch/font-ttf-dejavu-sans-mono-2.37-hab24e00_0.tar.bz2
+ - conda: https://conda.anaconda.org/conda-forge/noarch/font-ttf-inconsolata-3.000-h77eed37_0.tar.bz2
+ - conda: https://conda.anaconda.org/conda-forge/noarch/font-ttf-source-code-pro-2.038-h77eed37_0.tar.bz2
+ - conda: https://conda.anaconda.org/conda-forge/noarch/font-ttf-ubuntu-0.83-h77eed37_3.conda
+ - conda: https://conda.anaconda.org/conda-forge/linux-64/fontconfig-2.18.3-h4db4eae_1.conda
+ - conda: https://conda.anaconda.org/conda-forge/noarch/fonts-conda-ecosystem-1-0.tar.bz2
+ - conda: https://conda.anaconda.org/conda-forge/noarch/fonts-conda-forge-1-hc364b38_1.conda
+ - conda: https://conda.anaconda.org/conda-forge/linux-64/fribidi-1.0.18-h7cc23a3_0.conda
+ - conda: https://conda.anaconda.org/conda-forge/linux-64/gcc_impl_linux-64-16.2.0-h9891192_7.conda
+ - conda: https://conda.anaconda.org/conda-forge/linux-64/gcc_linux-64-16.2.0-h0ab548f_2.conda
+ - conda: https://conda.anaconda.org/conda-forge/linux-64/gfortran_impl_linux-64-16.2.0-hf930d89_7.conda
+ - conda: https://conda.anaconda.org/conda-forge/linux-64/graphite2-1.3.15-h54a6638_1.conda
+ - conda: https://conda.anaconda.org/conda-forge/linux-64/gsl-2.7-he838d99_0.tar.bz2
+ - conda: https://conda.anaconda.org/conda-forge/linux-64/gxx_impl_linux-64-16.2.0-h0d273dc_7.conda
+ - conda: https://conda.anaconda.org/conda-forge/linux-64/gxx_linux-64-16.2.0-h23aca61_2.conda
+ - conda: https://conda.anaconda.org/bioconda/linux-64/htslib-1.24-ha79157c_0.conda
+ - conda: https://conda.anaconda.org/conda-forge/linux-64/icu-78.3-py310h44b86e0_2.conda
+ - conda: https://conda.anaconda.org/conda-forge/noarch/kernel-headers_linux-64-4.18.0-he073ed8_9.conda
+ - conda: https://conda.anaconda.org/conda-forge/linux-64/keyutils-1.6.3-h7cc23a3_1.conda
+ - conda: https://conda.anaconda.org/conda-forge/linux-64/krb5-1.22.2-hbc21106_2.conda
+ - conda: https://conda.anaconda.org/conda-forge/linux-64/ld_impl_linux-64-2.46.1-default_hbd61a6d_103.conda
+ - conda: https://conda.anaconda.org/conda-forge/linux-64/lerc-4.3.0-he78707d_0.conda
+ - conda: https://conda.anaconda.org/conda-forge/linux-64/libblas-3.11.0-12_h4a7cf45_openblas.conda
+ - conda: https://conda.anaconda.org/conda-forge/linux-64/libcblas-3.11.0-12_h0358290_openblas.conda
+ - conda: https://conda.anaconda.org/conda-forge/linux-64/libcurl-8.23.0-ha042cf0_0.conda
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+ - conda: https://conda.anaconda.org/conda-forge/linux-64/libexpat-2.9.0-hd2095e1_0.conda
+ - conda: https://conda.anaconda.org/conda-forge/linux-64/libffi-3.7.0-h81df57d_1.conda
+ - conda: https://conda.anaconda.org/conda-forge/linux-64/libfreetype-2.14.3-ha770c72_2.conda
+ - conda: https://conda.anaconda.org/conda-forge/linux-64/libfreetype6-2.14.3-h5e6c136_2.conda
+ - conda: https://conda.anaconda.org/conda-forge/linux-64/libgcc-16.2.0-ha9f2e26_7.conda
+ - conda: https://conda.anaconda.org/conda-forge/noarch/libgcc-devel_linux-64-16.2.0-he3ce08f_107.conda
+ - conda: https://conda.anaconda.org/conda-forge/linux-64/libgcc-ng-16.2.0-h69a702a_7.conda
+ - conda: https://conda.anaconda.org/conda-forge/linux-64/libgfortran-16.2.0-h69a702a_7.conda
+ - conda: https://conda.anaconda.org/conda-forge/linux-64/libgfortran5-16.2.0-h6b99dfc_7.conda
+ - conda: https://conda.anaconda.org/conda-forge/linux-64/libglib-2.90.0-h569388d_0.conda
+ - conda: https://conda.anaconda.org/conda-forge/linux-64/libgomp-16.2.0-he0feb66_7.conda
+ - conda: https://conda.anaconda.org/conda-forge/linux-64/libharfbuzz-15.0.0-h23af247_0.conda
+ - conda: https://conda.anaconda.org/conda-forge/linux-64/libiconv-1.18-h0cb94f2_3.conda
+ - conda: https://conda.anaconda.org/conda-forge/linux-64/libjpeg-turbo-3.2.1-h7cc23a3_0.conda
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new file mode 100644
index 000000000000..e70903a7e234
--- /dev/null
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diff --git a/modules/nf-core/rastair2/mbias/environment.yml b/modules/nf-core/rastair2/mbias/environment.yml
new file mode 100644
index 000000000000..852efa2bba81
--- /dev/null
+++ b/modules/nf-core/rastair2/mbias/environment.yml
@@ -0,0 +1,10 @@
+---
+# yaml-language-server: $schema=https://raw.githubusercontent.com/nf-core/modules/master/modules/environment-schema.json
+channels:
+ - conda-forge
+ - bioconda
+dependencies:
+ # renovate: datasource=conda depName=bioconda/htslib
+ - bioconda::htslib=1.24
+ # renovate: datasource=conda depName=bioconda/rastair
+ - bioconda::rastair=2.2.0
diff --git a/modules/nf-core/rastair2/mbias/main.nf b/modules/nf-core/rastair2/mbias/main.nf
new file mode 100644
index 000000000000..20f67caef012
--- /dev/null
+++ b/modules/nf-core/rastair2/mbias/main.nf
@@ -0,0 +1,69 @@
+process RASTAIR2_MBIAS {
+ tag "${meta.id}"
+ label 'process_medium'
+
+ conda "${moduleDir}/environment.yml"
+ container "${workflow.containerEngine in ['singularity', 'apptainer'] && !task.ext.singularity_pull_docker_container
+ ? 'https://community-cr-prod.seqera.io/docker/registry/v2/blobs/sha256/50/50c87ecbec7ea76f99ff4ad84e16f083c0b74651bf813d9158b1004234f6feca/data'
+ : 'community.wave.seqera.io/library/htslib_rastair:4ffd26b40813e268'}"
+
+ input:
+ tuple val(meta), path(bam), path(index)
+ tuple val(meta2), path(fasta), path(fai)
+
+ output:
+ tuple val(meta), path("*.mbias.html"), emit: html
+ tuple val(meta), path("*.cutoffs.txt"), emit: cutoffs, optional: true
+ tuple val("${task.process}"), val('rastair'), eval("rastair --version | sed 's/^rastair //'"), emit: versions_rastair, topic: versions
+
+ when:
+ task.ext.when == null || task.ext.when
+
+ script:
+ def args = task.ext.args ?: ''
+ def args2 = task.ext.args2 ?: ''
+ def prefix = task.ext.prefix ?: "${meta.id}"
+ // The report tells read 1 from read 2 and OT from OB with read-pair flags, so it needs paired-end reads.
+ if (meta.single_end) {
+ error("rastair2 mbias needs paired-end reads: it assigns strands and read ends from read-pair flags")
+ }
+ // `rastair mbias --bam` resolves symlinks and misses the staged .bai/.fai, so per-read runs here
+ """
+ rastair per-read \\
+ ${args} \\
+ --threads ${task.cpus} \\
+ --fasta-file ${fasta} \\
+ --bed ${prefix}.per_read.bed.gz \\
+ ${bam}
+
+ # The report fails on an empty table, so reads without CpGs mean no estimate rather than an error
+ reads_with_cpgs=\$(gzip -dc ${prefix}.per_read.bed.gz | awk -F '\\t' '!/^#/ && \$10 > 0 { n++ } END { print n + 0 }')
+ if [ "\$reads_with_cpgs" -gt 0 ]; then
+ # rastair looks for its R scripts next to the binary, but conda installs them under share/
+ rastair mbias \\
+ ${args2} \\
+ --r-script-dir \$(dirname \$(which rastair))/../share/rastair/scripts \\
+ --no-vbias \\
+ --no-gc \\
+ --threads ${task.cpus} \\
+ --bed ${prefix}.per_read.bed.gz \\
+ --output-prefix .
+ mv qc_report.html ${prefix}.mbias.html
+ else
+ echo "${prefix}: no reads with CpGs in range, so no M-bias estimate.
" > ${prefix}.mbias.html
+ fi
+
+ rm ${prefix}.per_read.bed.gz ${prefix}.per_read.bed.gz.tbi
+ for cutoffs in *_cutoffs.txt; do
+ [ -e "\$cutoffs" ] || continue
+ mv "\$cutoffs" "${prefix}.\${cutoffs%_cutoffs.txt}.cutoffs.txt"
+ done
+ """
+
+ stub:
+ def prefix = task.ext.prefix ?: "${meta.id}"
+ """
+ touch ${prefix}.mbias.html
+ touch ${prefix}.chr1.cutoffs.txt
+ """
+}
diff --git a/modules/nf-core/rastair2/mbias/meta.yml b/modules/nf-core/rastair2/mbias/meta.yml
new file mode 100644
index 000000000000..f68e4b984b1a
--- /dev/null
+++ b/modules/nf-core/rastair2/mbias/meta.yml
@@ -0,0 +1,151 @@
+name: "rastair2_mbias"
+description: |
+ Measure methylation by position in read (M-bias) of 5mC->T converted
+ paired-end reads with rastair version 2, and suggest how many bases to
+ ignore at each read end. Runs `rastair per-read` (options in `ext.args`,
+ e.g. `--region`) and then `rastair mbias` on its output (options in
+ `ext.args2`). Writes an HTML report and, for each contig with enough data,
+ a cutoffs file to pass to rastair2/call.
+ Non-commercial licence: see the rastair tool description.
+keywords:
+ - methylation
+ - mbias
+ - taps
+ - 5-base
+ - qc
+ - trimming
+tools:
+ - "rastair":
+ description: |
+ Simultaneous detection of genetic variants and methylated positions from
+ TAPS+, Illumina 5-base or other 5mC->T converted sequencing data.
+ Licensing: rastair version 2 and newer is free for non-commercial research
+ use only. Commercial use (including providing paid services or developing
+ products) requires a licence from Oxford University Innovation
+ (enquiries@innovation.ox.ac.uk, reference 24811). Run `rastair license` for
+ the full terms. Publications using results must include the notice
+ "Copyright © 2025, University of Oxford".
+ homepage: "https://www.rastair.com"
+ documentation: "https://docs.rastair.com"
+ tool_dev_url: "https://github.com/bsbludwig/rastair"
+ licence: ["LicenseRef-NonCommercial"]
+ identifier: ""
+input:
+ - - meta:
+ type: map
+ description: |
+ Groovy Map containing sample information. Reads must be paired-end:
+ `single_end: true` is an error.
+ e.g. `[ id:'sample1', single_end:false ]`
+ - bam:
+ type: file
+ description: Coordinate-sorted BAM or CRAM file of 5mC->T converted paired-end reads
+ pattern: "*.{bam,cram}"
+ ontologies:
+ - edam: "http://edamontology.org/format_2572"
+ - edam: "http://edamontology.org/format_3462"
+ - index:
+ type: file
+ description: Index of the BAM or CRAM file
+ pattern: "*.{bai,csi,crai}"
+ ontologies:
+ - edam: "http://edamontology.org/format_3327"
+ - - meta2:
+ type: map
+ description: |
+ Groovy Map containing reference information
+ e.g. `[ id:'genome' ]`
+ - fasta:
+ type: file
+ description: Reference genome FASTA, used by `rastair per-read`
+ pattern: "*.{fa,fasta,fna}{,.gz}"
+ ontologies:
+ - edam: "http://edamontology.org/format_1929"
+ - fai:
+ type: file
+ description: |
+ samtools faidx index of the reference FASTA. Staged next to the FASTA,
+ where rastair finds it; it is not passed on the command line.
+ pattern: "*.fai"
+ ontologies:
+ - edam: "http://edamontology.org/format_3475"
+output:
+ html:
+ - - meta:
+ type: map
+ description: Groovy Map containing sample information. e.g. `[ id:'sample1' ]`
+ - "*.mbias.html":
+ type: file
+ description: |
+ HTML report with M-bias plots per contig and the suggested cutoffs drawn
+ on them. V-bias and GC plots are not produced.
+ pattern: "*.mbias.html"
+ ontologies:
+ - edam: "http://edamontology.org/format_2331"
+ cutoffs:
+ - - meta:
+ type: map
+ description: Groovy Map containing sample information. e.g. `[ id:'sample1' ]`
+ - "*.cutoffs.txt":
+ type: file
+ description: |
+ One file per contig, `..cutoffs.txt`. Line 1 is the value
+ for rastair2/call `--nOT`, line 2 for `--nOB`, each as
+ `r1_start,r1_end,r2_start,r2_end` bases to ignore. Restrict the run to one
+ contig or region with `--region` in `ext.args` to get a single file:
+ this also keeps memory down, as the report loads every read in range.
+ Contigs with too little data get no file, and if no read in range has a
+ CpG there are no cutoffs files at all (the report says so), so callers
+ can fall back to no trimming.
+ pattern: "*.cutoffs.txt"
+ ontologies:
+ - edam: "http://edamontology.org/format_3475"
+ versions_rastair:
+ - - ${task.process}:
+ type: string
+ description: The name of the process
+ - rastair:
+ type: string
+ description: The name of the tool
+ - "rastair --version | sed 's/^rastair //'":
+ type: eval
+ description: The expression to obtain the version of the tool
+topics:
+ versions:
+ - - ${task.process}:
+ type: string
+ description: The name of the process
+ - rastair:
+ type: string
+ description: The name of the tool
+ - "rastair --version | sed 's/^rastair //'":
+ type: eval
+ description: The expression to obtain the version of the tool
+authors:
+ - "@eduard-watchmaker"
+maintainers:
+ - "@eduard-watchmaker"
+containers:
+ docker:
+ linux/amd64:
+ name: community.wave.seqera.io/library/htslib_rastair:4ffd26b40813e268
+ build_id: bd-4ffd26b40813e268_1
+ scan_id: sc-b1009c453e710340_1
+ linux/arm64:
+ name: community.wave.seqera.io/library/htslib_rastair:0f7189390ccfdc4d
+ build_id: bd-0f7189390ccfdc4d_1
+ scan_id: sc-68e9105b71b20e61_1
+ singularity:
+ linux/amd64:
+ name: oras://community.wave.seqera.io/library/htslib_rastair:8f58b5250bf317c8
+ build_id: bd-8f58b5250bf317c8_1
+ https: https://community-cr-prod.seqera.io/docker/registry/v2/blobs/sha256/50/50c87ecbec7ea76f99ff4ad84e16f083c0b74651bf813d9158b1004234f6feca/data
+ linux/arm64:
+ name: oras://community.wave.seqera.io/library/htslib_rastair:42892508b9c8b619
+ build_id: bd-42892508b9c8b619_1
+ https: https://community-cr-prod.seqera.io/docker/registry/v2/blobs/sha256/11/11261c0ef97d532effc21421bd7c824749330dba152db93e4d5d583c362e0620/data
+ conda:
+ linux/amd64:
+ lock_file: modules/nf-core/rastair2/mbias/.conda-lock/linux_amd64-bd-4ffd26b40813e268_1.txt
+ linux/arm64:
+ lock_file: modules/nf-core/rastair2/mbias/.conda-lock/linux_arm64-bd-0f7189390ccfdc4d_1.txt
diff --git a/modules/nf-core/rastair2/mbias/tests/main.nf.test b/modules/nf-core/rastair2/mbias/tests/main.nf.test
new file mode 100644
index 000000000000..2a9cfdc0af00
--- /dev/null
+++ b/modules/nf-core/rastair2/mbias/tests/main.nf.test
@@ -0,0 +1,180 @@
+nextflow_process {
+
+ name "Test Process RASTAIR2_MBIAS"
+ script "../main.nf"
+ process "RASTAIR2_MBIAS"
+ config "./nextflow.config"
+
+ tag "modules"
+ tag "modules_nfcore"
+ tag "rastair2"
+ tag "rastair2/mbias"
+
+ // NOTE The HTML report embeds plots and run details, so only its name is snapshotted.
+
+ test("sarscov2 - paired-end methylated bam") {
+
+ when {
+ params {
+ module_args = ''
+ }
+ process {
+ """
+ input[0] = channel.of([
+ [ id:'test', single_end:false ],
+ file(params.modules_testdata_base_path + 'genomics/sarscov2/illumina/bam/test.paired_end.methylated.sorted.bam', checkIfExists: true),
+ file(params.modules_testdata_base_path + 'genomics/sarscov2/illumina/bam/test.paired_end.methylated.sorted.bam.bai', checkIfExists: true)
+ ])
+ input[1] = channel.of([
+ [ id:'genome' ],
+ file(params.modules_testdata_base_path + 'genomics/sarscov2/genome/genome.fasta', checkIfExists: true),
+ file(params.modules_testdata_base_path + 'genomics/sarscov2/genome/genome.fasta.fai', checkIfExists: true)
+ ])
+ """
+ }
+ }
+
+ then {
+ assert process.success
+ assertAll(
+ { assert path(process.out.html[0][1]).text.contains('M-bias') },
+ { assert snapshot(
+ process.out.html.collect { meta, html -> file(html).name },
+ process.out.cutoffs,
+ process.out.findAll { key, val -> key.startsWith("versions") }
+ ).match() }
+ )
+ }
+ }
+
+ test("sarscov2 - paired-end methylated bam - region") {
+
+ when {
+ params {
+ module_args = '--region MT192765.1:1-15000'
+ }
+ process {
+ """
+ input[0] = channel.of([
+ [ id:'test', single_end:false ],
+ file(params.modules_testdata_base_path + 'genomics/sarscov2/illumina/bam/test.paired_end.methylated.sorted.bam', checkIfExists: true),
+ file(params.modules_testdata_base_path + 'genomics/sarscov2/illumina/bam/test.paired_end.methylated.sorted.bam.bai', checkIfExists: true)
+ ])
+ input[1] = channel.of([
+ [ id:'genome' ],
+ file(params.modules_testdata_base_path + 'genomics/sarscov2/genome/genome.fasta', checkIfExists: true),
+ file(params.modules_testdata_base_path + 'genomics/sarscov2/genome/genome.fasta.fai', checkIfExists: true)
+ ])
+ """
+ }
+ }
+
+ then {
+ assert process.success
+ assertAll(
+ { assert snapshot(
+ process.out.html.collect { meta, html -> file(html).name },
+ process.out.cutoffs,
+ process.out.findAll { key, val -> key.startsWith("versions") }
+ ).match() }
+ )
+ }
+ }
+
+ test("sarscov2 - paired-end methylated bam - no reads in range") {
+
+ when {
+ params {
+ module_args = '--min-mapq 255'
+ }
+ process {
+ """
+ input[0] = channel.of([
+ [ id:'test', single_end:false ],
+ file(params.modules_testdata_base_path + 'genomics/sarscov2/illumina/bam/test.paired_end.methylated.sorted.bam', checkIfExists: true),
+ file(params.modules_testdata_base_path + 'genomics/sarscov2/illumina/bam/test.paired_end.methylated.sorted.bam.bai', checkIfExists: true)
+ ])
+ input[1] = channel.of([
+ [ id:'genome' ],
+ file(params.modules_testdata_base_path + 'genomics/sarscov2/genome/genome.fasta', checkIfExists: true),
+ file(params.modules_testdata_base_path + 'genomics/sarscov2/genome/genome.fasta.fai', checkIfExists: true)
+ ])
+ """
+ }
+ }
+
+ then {
+ assert process.success
+ assertAll(
+ { assert process.out.cutoffs.size() == 0 },
+ { assert snapshot(
+ process.out.html,
+ process.out.findAll { key, val -> key.startsWith("versions") }
+ ).match() }
+ )
+ }
+ }
+
+ test("sarscov2 - single-end bam - fails") {
+
+ when {
+ params {
+ module_args = ''
+ }
+ process {
+ """
+ input[0] = channel.of([
+ [ id:'test', single_end:true ],
+ file(params.modules_testdata_base_path + 'genomics/sarscov2/illumina/bam/test.single_end.sorted.bam', checkIfExists: true),
+ file(params.modules_testdata_base_path + 'genomics/sarscov2/illumina/bam/test.single_end.sorted.bam.bai', checkIfExists: true)
+ ])
+ input[1] = channel.of([
+ [ id:'genome' ],
+ file(params.modules_testdata_base_path + 'genomics/sarscov2/genome/genome.fasta', checkIfExists: true),
+ file(params.modules_testdata_base_path + 'genomics/sarscov2/genome/genome.fasta.fai', checkIfExists: true)
+ ])
+ """
+ }
+ }
+
+ then {
+ assertAll(
+ { assert process.failed },
+ { assert process.errorReport.contains('rastair2 mbias needs paired-end reads') }
+ )
+ }
+ }
+
+ test("sarscov2 - paired-end methylated bam - stub") {
+
+ options "-stub"
+
+ when {
+ params {
+ module_args = ''
+ }
+ process {
+ """
+ input[0] = channel.of([
+ [ id:'test', single_end:false ],
+ file(params.modules_testdata_base_path + 'genomics/sarscov2/illumina/bam/test.paired_end.methylated.sorted.bam', checkIfExists: true),
+ file(params.modules_testdata_base_path + 'genomics/sarscov2/illumina/bam/test.paired_end.methylated.sorted.bam.bai', checkIfExists: true)
+ ])
+ input[1] = channel.of([
+ [ id:'genome' ],
+ file(params.modules_testdata_base_path + 'genomics/sarscov2/genome/genome.fasta', checkIfExists: true),
+ file(params.modules_testdata_base_path + 'genomics/sarscov2/genome/genome.fasta.fai', checkIfExists: true)
+ ])
+ """
+ }
+ }
+
+ then {
+ assert process.success
+ assertAll(
+ { assert snapshot(sanitizeOutput(process.out)).match() }
+ )
+ }
+ }
+
+}
diff --git a/modules/nf-core/rastair2/mbias/tests/main.nf.test.snap b/modules/nf-core/rastair2/mbias/tests/main.nf.test.snap
new file mode 100644
index 000000000000..e12286403233
--- /dev/null
+++ b/modules/nf-core/rastair2/mbias/tests/main.nf.test.snap
@@ -0,0 +1,125 @@
+{
+ "sarscov2 - paired-end methylated bam": {
+ "content": [
+ [
+ "test.mbias.html"
+ ],
+ [
+ [
+ {
+ "id": "test",
+ "single_end": false
+ },
+ "test.MT192765.1.cutoffs.txt:md5,e46ba9ffc77f03688559a3007e005d91"
+ ]
+ ],
+ {
+ "versions_rastair": [
+ [
+ "RASTAIR2_MBIAS",
+ "rastair",
+ "2.2.0"
+ ]
+ ]
+ }
+ ],
+ "timestamp": "2026-11-10T10:59:21.558524",
+ "meta": {
+ "nf-test": "0.9.5",
+ "nextflow": "26.10.5"
+ }
+ },
+ "sarscov2 - paired-end methylated bam - region": {
+ "content": [
+ [
+ "test.mbias.html"
+ ],
+ [
+ [
+ {
+ "id": "test",
+ "single_end": false
+ },
+ "test.MT192765.1.cutoffs.txt:md5,e46ba9ffc77f03688559a3007e005d91"
+ ]
+ ],
+ {
+ "versions_rastair": [
+ [
+ "RASTAIR2_MBIAS",
+ "rastair",
+ "2.2.0"
+ ]
+ ]
+ }
+ ],
+ "timestamp": "2026-11-10T10:59:35.019163",
+ "meta": {
+ "nf-test": "0.9.5",
+ "nextflow": "26.10.5"
+ }
+ },
+ "sarscov2 - paired-end methylated bam - stub": {
+ "content": [
+ {
+ "cutoffs": [
+ [
+ {
+ "id": "test",
+ "single_end": false
+ },
+ "test.chr1.cutoffs.txt:md5,d41d8cd98f00b204e9800998ecf8427e"
+ ]
+ ],
+ "html": [
+ [
+ {
+ "id": "test",
+ "single_end": false
+ },
+ "test.mbias.html:md5,d41d8cd98f00b204e9800998ecf8427e"
+ ]
+ ],
+ "versions_rastair": [
+ [
+ "RASTAIR2_MBIAS",
+ "rastair",
+ "2.2.0"
+ ]
+ ]
+ }
+ ],
+ "timestamp": "2026-11-10T10:59:53.199379",
+ "meta": {
+ "nf-test": "0.9.5",
+ "nextflow": "26.10.5"
+ }
+ },
+ "sarscov2 - paired-end methylated bam - no reads in range": {
+ "content": [
+ [
+ [
+ {
+ "id": "test",
+ "single_end": false
+ },
+ "test.mbias.html:md5,79d7ceabea7359ea6c4fc550d71089ba"
+ ]
+ ],
+ {
+ "versions_rastair": [
+ [
+ "RASTAIR2_MBIAS",
+ "rastair",
+ "2.2.0"
+ ]
+ ]
+ }
+ ],
+ "timestamp": "2026-11-10T12:02:56.408085",
+ "meta": {
+ "nf-test": "0.9.5",
+ "nextflow": "26.10.5"
+ }
+ }
+}
\ No newline at end of file
diff --git a/modules/nf-core/rastair2/mbias/tests/nextflow.config b/modules/nf-core/rastair2/mbias/tests/nextflow.config
new file mode 100644
index 000000000000..703c2e86f1cb
--- /dev/null
+++ b/modules/nf-core/rastair2/mbias/tests/nextflow.config
@@ -0,0 +1,5 @@
+process {
+ withName: 'RASTAIR2_MBIAS' {
+ ext.args = params.module_args
+ }
+}