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main.nf
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#!/usr/bin/env nextflow
/*
========================================================================================
nf-core/methylseq
========================================================================================
nf-core/methylseq Analysis Pipeline.
#### Homepage / Documentation
https://github.com/nf-core/methylseq
----------------------------------------------------------------------------------------
*/
log.info Headers.nf_core(workflow, params.monochrome_logs)
////////////////////////////////////////////////////
/* -- PRINT HELP -- */
////////////////////////////////////////////////////+
def json_schema = "$projectDir/nextflow_schema.json"
if (params.help) {
def command = "nextflow run nf-core/methylseq --input '*_R{1,2}.fastq.gz' -profile docker"
log.info NfcoreSchema.params_help(workflow, params, json_schema, command)
exit 0
}
////////////////////////////////////////////////////
/* -- VALIDATE PARAMETERS -- */
////////////////////////////////////////////////////+
if (params.validate_params) {
NfcoreSchema.validateParameters(params, json_schema, log)
}
////////////////////////////////////////////////////
/* -- Collect configuration parameters -- */
////////////////////////////////////////////////////
// These params need to be set late, after the iGenomes config is loaded
params.bismark_index = params.genome ? params.genomes[ params.genome ].bismark ?: false : false
params.bwa_meth_index = params.genome ? params.genomes[ params.genome ].bwa_meth ?: false : false
params.fasta = params.genome ? params.genomes[ params.genome ].fasta ?: false : false
params.fasta_index = params.genome ? params.genomes[ params.genome ].fasta_index ?: false : false
// Check if genome exists in the config file
if (params.genomes && params.genome && !params.genomes.containsKey(params.genome)) {
exit 1, "The provided genome '${params.genome}' is not available in the iGenomes file. Currently the available genomes are ${params.genomes.keySet().join(', ')}"
}
Channel
.fromPath("$projectDir/assets/where_are_my_files.txt", checkIfExists: true)
.into { ch_wherearemyfiles_for_trimgalore; ch_wherearemyfiles_for_alignment }
ch_splicesites_for_bismark_hisat_align = params.known_splices ? Channel.fromPath(params.known_splices, checkIfExists: true) : Channel.empty()
if( params.aligner =~ /bismark/ ){
assert params.bismark_index || params.fasta : "No reference genome index or fasta file specified"
ch_wherearemyfiles_for_alignment.set { ch_wherearemyfiles_for_bismark_align }
if( params.bismark_index ){
Channel
.fromPath(params.bismark_index, checkIfExists: true)
.ifEmpty { exit 1, "Bismark index file not found: ${params.bismark_index}" }
.into { ch_bismark_index_for_bismark_align; ch_bismark_index_for_bismark_methXtract }
}
else if( params.fasta ){
Channel
.fromPath(params.fasta, checkIfExists: true)
.ifEmpty { exit 1, "fasta file not found : ${params.fasta}" }
.set { ch_fasta_for_makeBismarkIndex }
}
}
else if( params.aligner == 'bwameth' ){
assert params.fasta : "No Fasta reference specified! This is required by MethylDackel."
ch_wherearemyfiles_for_alignment.into { ch_wherearemyfiles_for_bwamem_align; ch_wherearemyfiles_for_samtools_sort_index_flagstat }
Channel
.fromPath(params.fasta, checkIfExists: true)
.ifEmpty { exit 1, "fasta file not found : ${params.fasta}" }
.into { ch_fasta_for_makeBwaMemIndex; ch_fasta_for_makeFastaIndex; ch_fasta_for_methyldackel }
if( params.bwa_meth_index ){
Channel
.fromPath("${params.bwa_meth_index}*", checkIfExists: true)
.ifEmpty { exit 1, "bwa-meth index file(s) not found: ${params.bwa_meth_index}" }
.set { ch_bwa_meth_indices_for_bwamem_align }
ch_fasta_for_makeBwaMemIndex.close()
}
if( params.fasta_index ){
Channel
.fromPath(params.fasta_index, checkIfExists: true)
.ifEmpty { exit 1, "fasta index file not found: ${params.fasta_index}" }
.set { ch_fasta_index_for_methyldackel }
ch_fasta_for_makeFastaIndex.close()
}
}
Channel
.fromPath(params.fasta, checkIfExists: true)
.ifEmpty { exit 1, "fasta file not found : ${params.fasta}" }
.into { ch_fasta_for_cgmaptools; ch_fasta_bismarkIndex_2 }
// Trimming / kit presets
clip_r1 = params.clip_r1
clip_r2 = params.clip_r2
three_prime_clip_r1 = params.three_prime_clip_r1
three_prime_clip_r2 = params.three_prime_clip_r2
bismark_minins = params.minins
bismark_maxins = params.maxins
if(params.pbat){
clip_r1 = 9
clip_r2 = 9
three_prime_clip_r1 = 9
three_prime_clip_r2 = 9
}
else if( params.single_cell ){
clip_r1 = 6
clip_r2 = 6
three_prime_clip_r1 = 6
three_prime_clip_r2 = 6
}
else if( params.epignome ){
clip_r1 = 8
clip_r2 = 8
three_prime_clip_r1 = 8
three_prime_clip_r2 = 8
}
else if( params.accel || params.zymo ){
clip_r1 = 10
clip_r2 = 15
three_prime_clip_r1 = 10
three_prime_clip_r2 = 10
}
else if( params.cegx ){
clip_r1 = 6
clip_r2 = 6
three_prime_clip_r1 = 2
three_prime_clip_r2 = 2
}
else if( params.em_seq ){
bismark_maxins = 1000
clip_r1 = 8
clip_r2 = 8
three_prime_clip_r1 = 8
three_prime_clip_r2 = 8
}
// Check AWS batch settings
if (workflow.profile.contains('awsbatch')) {
// AWSBatch sanity checking
if (!params.awsqueue || !params.awsregion) exit 1, 'Specify correct --awsqueue and --awsregion parameters on AWSBatch!'
// Check outdir paths to be S3 buckets if running on AWSBatch
// related: https://github.com/nextflow-io/nextflow/issues/813
if (!params.outdir.startsWith('s3:')) exit 1, 'Outdir not on S3 - specify S3 Bucket to run on AWSBatch!'
// Prevent trace files to be stored on S3 since S3 does not support rolling files.
if (params.tracedir.startsWith('s3:')) exit 1, 'Specify a local tracedir or run without trace! S3 cannot be used for tracefiles.'
}
// Stage config files
ch_multiqc_config = file("$projectDir/assets/multiqc_config.yaml", checkIfExists: true)
ch_multiqc_custom_config = params.multiqc_config ? Channel.fromPath(params.multiqc_config, checkIfExists: true) : Channel.empty()
ch_output_docs = file("$projectDir/docs/output.md", checkIfExists: true)
ch_output_docs_images = file("$projectDir/docs/images/", checkIfExists: true)
/*
* Create a channel for input read files
*/
if (params.input_paths) {
if (params.single_end) {
Channel
.from(params.input_paths)
.map { row -> [ row[0], [ file(row[1][0], checkIfExists: true) ] ] }
.ifEmpty { exit 1, 'params.input_paths was empty - no input files supplied' }
.into { ch_read_files_fastqc; ch_read_files_trimming }
} else {
Channel
.from(params.input_paths)
.map { row -> [ row[0], [ file(row[1][0], checkIfExists: true), file(row[1][1], checkIfExists: true) ] ] }
.ifEmpty { exit 1, 'params.input_paths was empty - no input files supplied' }
.into { ch_read_files_fastqc; ch_read_files_trimming }
}
}
else if (params.aligner == 'none'){
Channel
.fromPath(params.bam)
.map { file -> tuple(file.baseName, file) }
.ifEmpty { exit 1, 'params.bam_paths was empty - no input files supplied' }
.set { ch_indep_bam_for_processing }
}
else {
Channel
.fromFilePairs(params.input, size: params.single_end ? 1 : 2)
.ifEmpty { exit 1, "Cannot find any reads matching: ${params.input}\nNB: Path needs to be enclosed in quotes!\nIf this is single-end data, please specify --single_end on the command line." }
.into { ch_read_files_fastqc; ch_read_files_trimming}
}
/* .map { path -> filename = path.getName()
if (filename.endsWith('.bam'))
filename = filename.substring(0, filename.length() - 3)
return [path, filename] } */
/* else if (params.aligner == 'none'){
Channel
.fromPath(params.bam)
.map { file -> tuple(file.baseName, file) }
.ifEmpty { exit 1, 'params.bam_paths was empty - no input files supplied' }
.set { ch_indep_bam_for_processing }
}
////////////////////////////////////////////////////
/* -- PRINT PARAMETER SUMMARY -- */
////////////////////////////////////////////////////
log.info NfcoreSchema.params_summary_log(workflow, params, json_schema)
// Header log info
def summary = [:]
if (workflow.revision) summary['Pipeline Release'] = workflow.revision
summary['Run Name'] = workflow.runName
summary['Aligner'] = params.aligner
summary['Data Type'] = params.single_end ? 'Single-End' : 'Paired-End'
if (params.input) summary['Input'] = params.input
if(params.known_splices) summary['Spliced alignment'] = 'Yes'
if(params.slamseq) summary['SLAM-seq'] = 'Yes'
if(params.local_alignment) summary['Local alignment'] = 'Yes'
if(params.genome) summary['Genome'] = params.genome
if(params.bismark_index) summary['Bismark Index'] = params.bismark_index
if(params.bwa_meth_index) summary['BWA-Meth Index'] = "${params.bwa_meth_index}*"
if(params.fasta) summary['Fasta Ref'] = params.fasta
if(params.bam) summary['Bam input'] = params.bam
if(params.fasta_index) summary['Fasta Index'] = params.fasta_index
if(params.rrbs) summary['RRBS Mode'] = 'On'
if(params.relax_mismatches) summary['Mismatch Func'] = "L,0,-${params.num_mismatches} (Bismark default = L,0,-0.2)"
if(params.skip_trimming) summary['Trimming Step'] = 'Skipped'
if(params.skip_alignment) summary['Trimming Step','FastQC step', 'Build index step', 'Alignment step'] = 'Skipped'
if(params.pbat) summary['Trim Profile'] = 'PBAT'
if(params.single_cell) summary['Trim Profile'] = 'Single Cell'
if(params.epignome) summary['Trim Profile'] = 'TruSeq (EpiGnome)'
if(params.accel) summary['Trim Profile'] = 'Accel-NGS (Swift)'
if(params.zymo) summary['Trim Profile'] = 'Zymo Pico-Methyl'
if(params.cegx) summary['Trim Profile'] = 'CEGX'
if(params.em_seq) summary['Trim Profile'] = 'EM Seq'
summary['Trimming'] = "5'R1: $clip_r1 / 5'R2: $clip_r2 / 3'R1: $three_prime_clip_r1 / 3'R2: $three_prime_clip_r2"
summary['Deduplication'] = params.skip_deduplication || params.rrbs ? 'No' : 'Yes'
summary['Directional Mode'] = params.single_cell || params.zymo || params.non_directional ? 'No' : 'Yes'
summary['All C Contexts'] = params.comprehensive ? 'Yes' : 'No'
summary['Cytosine report'] = params.cytosine_report ? 'Yes' : 'No'
if(params.min_depth) summary['Minimum Depth'] = params.min_depth
if(params.ignore_flags) summary['MethylDackel'] = 'Ignoring SAM Flags'
if(params.methyl_kit) summary['MethylDackel'] = 'Producing methyl_kit output'
save_intermeds = [];
if(params.save_reference) save_intermeds.add('Reference genome build')
if(params.save_trimmed) save_intermeds.add('Trimmed FastQ files')
if(params.unmapped) save_intermeds.add('Unmapped reads')
if(params.save_align_intermeds) save_intermeds.add('Intermediate BAM files')
if(save_intermeds.size() > 0) summary['Save Intermediates'] = save_intermeds.join(', ')
if(params.minins) summary['Bismark min insert size'] = bismark_minins
if(params.maxins || params.em_seq) summary['Bismark max insert size'] = bismark_maxins
if(params.bismark_align_cpu_per_multicore) summary['Bismark align CPUs per --multicore'] = params.bismark_align_cpu_per_multicore
if(params.bismark_align_mem_per_multicore) summary['Bismark align memory per --multicore'] = params.bismark_align_mem_per_multicore
summary['Output dir'] = params.outdir
summary['Launch dir'] = workflow.launchDir
summary['Working dir'] = workflow.workDir
summary['Pipeline dir'] = workflow.projectDir
summary['User'] = workflow.userName
summary['Config Profile'] = workflow.profile
if (workflow.containerEngine) summary['Container'] = "$workflow.containerEngine - $workflow.container"
if (workflow.profile.contains('awsbatch')) {
summary['AWS Region'] = params.awsregion
summary['AWS Queue'] = params.awsqueue
summary['AWS CLI'] = params.awscli
}
summary['Max Resources'] = "$params.max_memory memory, $params.max_cpus cpus, $params.max_time time per job"
summary['Config Profile'] = workflow.profile
if (params.config_profile_description) summary['Config Profile Description'] = params.config_profile_description
if (params.config_profile_contact) summary['Config Profile Contact'] = params.config_profile_contact
if (params.config_profile_url) summary['Config Profile URL'] = params.config_profile_url
summary['Config Files'] = workflow.configFiles.join(', ')
if (params.email || params.email_on_fail) {
summary['E-mail Address'] = params.email
summary['E-mail on failure'] = params.email_on_fail
summary['MultiQC maxsize'] = params.max_multiqc_email_size
}
// Check that --project is set for the UPPMAX cluster
if( workflow.profile.contains('uppmax') ){
if( !params.project ) exit 1, "No UPPMAX project ID found! Use --project"
summary['Cluster Project'] = params.project
}
// Check the hostnames against configured profiles
checkHostname()
Channel.from(summary.collect{ [it.key, it.value] })
.map { k,v -> "<dt>$k</dt><dd><samp>${v ?: '<span style=\"color:#999999;\">N/A</a>'}</samp></dd>" }
.reduce { a, b -> return [a, b].join("\n ") }
.map { x -> """
id: 'nf-core-methylseq-summary'
description: " - this information is collected when the pipeline is started."
section_name: 'nf-core/methylseq Workflow Summary'
section_href: 'https://github.com/nf-core/methylseq'
plot_type: 'html'
data: |
<dl class=\"dl-horizontal\">
$x
</dl>
""".stripIndent() }
.set { ch_workflow_summary }
/*
* Parse software version numbers
*/
process get_software_versions {
publishDir "${params.outdir}/pipeline_info", mode: params.publish_dir_mode,
saveAs: { filename ->
if (filename.indexOf('.csv') > 0) filename
else null
}
output:
file 'software_versions_mqc.yaml' into ch_software_versions_yaml_for_multiqc
file "software_versions.csv"
script:
"""
echo "$workflow.manifest.version" &> v_pipeline.txt
echo "$workflow.nextflow.version" &> v_nextflow.txt
bismark_genome_preparation --version &> v_bismark_genome_preparation.txt
fastqc --version &> v_fastqc.txt
cutadapt --version &> v_cutadapt.txt
trim_galore --version &> v_trim_galore.txt
bismark --version &> v_bismark.txt
deduplicate_bismark --version &> v_deduplicate_bismark.txt
bismark_methylation_extractor --version &> v_bismark_methylation_extractor.txt
bismark2report --version &> v_bismark2report.txt
bismark2summary --version &> v_bismark2summary.txt
samtools --version &> v_samtools.txt
hisat2 --version &> v_hisat2.txt
bwa &> v_bwa.txt 2>&1 || true
bwameth.py --version &> v_bwameth.txt
picard MarkDuplicates --version &> v_picard_markdups.txt 2>&1 || true
MethylDackel --version &> v_methyldackel.txt
qualimap --version &> v_qualimap.txt || true
preseq &> v_preseq.txt
multiqc --version &> v_multiqc.txt
scrape_software_versions.py &> software_versions_mqc.yaml
"""
}
/*
* PREPROCESSING - Build Bismark index
*/
if( !params.bismark_index && params.aligner =~ /bismark/ ){
process makeBismarkIndex {
publishDir path: { params.save_reference ? "${params.outdir}/reference_genome" : params.outdir },
saveAs: { params.save_reference ? it : null }, mode: params.publish_dir_mode
input:
file fasta from ch_fasta_for_makeBismarkIndex
output:
file "BismarkIndex" into ch_bismark_index_for_bismark_align, ch_bismark_index_for_bismark_methXtract , ch_bismark_index_for_bismark_methXtract_2
script:
aligner = params.aligner == 'bismark_hisat' ? '--hisat2' : '--bowtie2'
slam = params.slamseq ? '--slam' : ''
"""
mkdir BismarkIndex
cp $fasta BismarkIndex/
bismark_genome_preparation $aligner $slam BismarkIndex
"""
}
}
/*
* PREPROCESSING - Build bwa-mem index
*/
if( !params.bwa_meth_index && params.aligner == 'bwameth' ){
process makeBwaMemIndex {
tag "$fasta"
publishDir path: "${params.outdir}/reference_genome", saveAs: { params.save_reference ? it : null }, mode: params.publish_dir_mode
input:
file fasta from ch_fasta_for_makeBwaMemIndex
output:
file "${fasta}*" into ch_bwa_meth_indices_for_bwamem_align
file fasta
script:
"""
bwameth.py index $fasta
"""
}
}
/*
* PREPROCESSING - Index Fasta file
*/
if( !params.fasta_index && params.aligner == 'bwameth' ){
process makeFastaIndex {
tag "$fasta"
publishDir path: "${params.outdir}/reference_genome", saveAs: { params.save_reference ? it : null }, mode: params.publish_dir_mode
input:
file fasta from ch_fasta_for_makeFastaIndex
output:
file "${fasta}.fai" into ch_fasta_index_for_methyldackel
script:
"""
samtools faidx $fasta
"""
}
}
/*
* STEP 1 - FastQC
*/
if( params.skip_alignment ){
ch_fastqc_results_for_multiqc = Channel.from(false)
} else {
process fastqc {
tag "$name"
label 'process_medium'
publishDir "${params.outdir}/fastqc", mode: params.publish_dir_mode,
saveAs: { filename ->
filename.indexOf('.zip') > 0 ? "zips/$filename" : "$filename"
}
input:
set val(name), file(reads) from ch_read_files_fastqc
output:
file '*_fastqc.{zip,html}' into ch_fastqc_results_for_multiqc
script:
"""
fastqc --quiet --threads $task.cpus $reads
"""
}
}
/*
* STEP 2 - Trim Galore!
*/
if( params.skip_trimming){
ch_trimmed_reads_for_alignment = ch_read_files_trimming
ch_trim_galore_results_for_multiqc = Channel.from(false)
}
else if (params.skip_alignment) {
ch_read_files_trimming = Channel.from(false)
ch_trim_galore_results_for_multiqc = Channel.from(false)
}
else {
process trim_galore {
tag "$name"
publishDir "${params.outdir}/trim_galore", mode: params.publish_dir_mode,
saveAs: {filename ->
if( filename.indexOf("_fastqc") > 0 ) "FastQC/$filename"
else if( filename.indexOf("trimming_report.txt" ) > 0) "logs/$filename"
else if( !params.save_trimmed && filename == "where_are_my_files.txt" ) filename
else if( params.save_trimmed && filename != "where_are_my_files.txt" ) filename
else null
}
input:
set val(name), file(reads) from ch_read_files_trimming
file wherearemyfiles from ch_wherearemyfiles_for_trimgalore.collect()
output:
set val(name), file('*fq.gz') into ch_trimmed_reads_for_alignment
file "*trimming_report.txt" into ch_trim_galore_results_for_multiqc
file "*_fastqc.{zip,html}"
file "where_are_my_files.txt"
script:
def c_r1 = clip_r1 > 0 ? "--clip_r1 $clip_r1" : ''
def c_r2 = clip_r2 > 0 ? "--clip_r2 $clip_r2" : ''
def tpc_r1 = three_prime_clip_r1 > 0 ? "--three_prime_clip_r1 $three_prime_clip_r1" : ''
def tpc_r2 = three_prime_clip_r2 > 0 ? "--three_prime_clip_r2 $three_prime_clip_r2" : ''
def rrbs = params.rrbs ? "--rrbs" : ''
def cores = 1
if(task.cpus){
cores = (task.cpus as int) - 4
if (params.single_end) cores = (task.cpus as int) - 3
if (cores < 1) cores = 1
if (cores > 4) cores = 4
}
if( params.single_end ) {
"""
trim_galore --fastqc --gzip $reads \
$rrbs $c_r1 $tpc_r1 --cores $cores
"""
} else {
"""
trim_galore --fastqc --gzip --paired $reads \
$rrbs $c_r1 $c_r2 $tpc_r1 $tpc_r2 --cores $cores
"""
}
}
}
/*
* STEP 3.1 - align with Bismark
*/
if( params.aligner =~ /bismark/ ){
process bismark_align {
tag "$name"
publishDir "${params.outdir}/bismark_alignments", mode: params.publish_dir_mode,
saveAs: {filename ->
if( filename.indexOf(".fq.gz") > 0 ) "unmapped/$filename"
else if( filename.indexOf("report.txt") > 0 ) "logs/$filename"
else if( (!params.save_align_intermeds && !params.skip_deduplication && !params.rrbs).every() && filename == "where_are_my_files.txt" ) filename
else if( (params.save_align_intermeds || params.skip_deduplication || params.rrbs).any() && filename != "where_are_my_files.txt" ) filename
else null
}
input:
set val(name), file(reads) from ch_trimmed_reads_for_alignment
file index from ch_bismark_index_for_bismark_align.collect()
file wherearemyfiles from ch_wherearemyfiles_for_bismark_align.collect()
file knownsplices from ch_splicesites_for_bismark_hisat_align.collect().ifEmpty([])
output:
set val(name), file("*.bam") into ch_bam_for_bismark_deduplicate, ch_bam_for_bismark_summary, ch_bam_for_preseq
set val(name), file("*report.txt") into ch_bismark_align_log_for_bismark_report, ch_bismark_align_log_for_bismark_summary, ch_bismark_align_log_for_multiqc
file "*.fq.gz" optional true
file "where_are_my_files.txt"
script:
// Paired-end or single end input files
input = params.single_end ? reads : "-1 ${reads[0]} -2 ${reads[1]}"
// Choice of read aligner
aligner = params.aligner == "bismark_hisat" ? "--hisat2" : "--bowtie2"
// Optional extra bismark parameters
splicesites = params.aligner == "bismark_hisat" && params.known_splices ? "--known-splicesite-infile <(hisat2_extract_splice_sites.py ${knownsplices})" : ''
pbat = params.pbat ? "--pbat" : ''
non_directional = params.single_cell || params.zymo || params.non_directional ? "--non_directional" : ''
unmapped = params.unmapped ? "--unmapped" : ''
mismatches = params.relax_mismatches ? "--score_min L,0,-${params.num_mismatches}" : ''
soft_clipping = params.local_alignment ? "--local" : ''
minins = bismark_minins ? "--minins $bismark_minins" : ''
maxins = bismark_maxins ? "--maxins $bismark_maxins" : ''
// Try to assign sensible bismark memory units according to what the task was given
multicore = ''
if( task.cpus ){
// Numbers based on recommendation by Felix for a typical mouse genome
if( params.single_cell || params.zymo || params.non_directional ){
cpu_per_multicore = 5
mem_per_multicore = (18.GB).toBytes()
} else {
cpu_per_multicore = 3
mem_per_multicore = (13.GB).toBytes()
}
// Check if the user has specified this and overwrite if so
if(params.bismark_align_cpu_per_multicore) {
cpu_per_multicore = (params.bismark_align_cpu_per_multicore as int)
}
if(params.bismark_align_mem_per_multicore) {
mem_per_multicore = (params.bismark_align_mem_per_multicore as nextflow.util.MemoryUnit).toBytes()
}
// How many multicore splits can we afford with the cpus we have?
ccore = ((task.cpus as int) / cpu_per_multicore) as int
// Check that we have enough memory, assuming 13GB memory per instance (typical for mouse alignment)
try {
tmem = (task.memory as nextflow.util.MemoryUnit).toBytes()
mcore = (tmem / mem_per_multicore) as int
ccore = Math.min(ccore, mcore)
} catch (all) {
log.debug "Warning: Not able to define bismark align multicore based on available memory"
}
if( ccore > 1 ){
multicore = "--multicore $ccore"
}
}
// Main command
"""
bismark $input \\
$aligner \\
--bam $pbat $non_directional $unmapped $mismatches $multicore $minins $maxins \\
--genome $index \\
$reads \\
$soft_clipping \\
$splicesites
"""
}
/*
* STEP 4 - Bismark deduplicate
*/
if( params.skip_deduplication || params.rrbs ) {
ch_bam_for_bismark_deduplicate.into { ch_bam_dedup_for_bismark_methXtract; ch_bam_dedup_for_qualimap; ch_bam_cgmaptools }
ch_bismark_dedup_log_for_bismark_report = Channel.from(false)
ch_bismark_dedup_log_for_bismark_summary = Channel.from(false)
ch_bismark_dedup_log_for_multiqc = Channel.from(false)
}
else{
process bismark_deduplicate {
tag "$name"
publishDir "${params.outdir}/bismark_deduplicated", mode: params.publish_dir_mode,
saveAs: {filename -> filename.indexOf(".bam") == -1 ? "logs/$filename" : "$filename"}
input:
set val(name), file(bam) from ch_bam_for_bismark_deduplicate
output:
set val(name), file("*.deduplicated.bam") into ch_bam_dedup_for_bismark_methXtract, ch_bam_dedup_for_qualimap, ch_bam_cgmaptools
set val(name), file("*.deduplication_report.txt") into ch_bismark_dedup_log_for_bismark_report, ch_bismark_dedup_log_for_bismark_summary, ch_bismark_dedup_log_for_multiqc
script:
fq_type = params.single_end ? '-s' : '-p'
"""
deduplicate_bismark $fq_type --bam $bam
"""
}
}
/*
* STEP 5 - Bismark methylation extraction
*/
process bismark_methXtract {
tag "$name"
publishDir "${params.outdir}/bismark_methylation_calls", mode: params.publish_dir_mode,
saveAs: {filename ->
if( filename.indexOf("splitting_report.txt" ) > 0 ) "logs/$filename"
else if( filename.indexOf("M-bias" ) > 0) "m-bias/$filename"
else if( filename.indexOf(".cov" ) > 0 ) "methylation_coverage/$filename"
else if( filename.indexOf("bedGraph" ) > 0 ) "bedGraph/$filename"
else if( filename.indexOf("CpG_report" ) > 0 ) "stranded_CpG_report/$filename"
else "methylation_calls/$filename"
}
input:
set val(name), file(bam) from ch_bam_dedup_for_bismark_methXtract
file index from ch_bismark_index_for_bismark_methXtract.collect()
output:
set val(name), file("*splitting_report.txt") into ch_bismark_splitting_report_for_bismark_report, ch_bismark_splitting_report_for_bismark_summary, ch_bismark_splitting_report_for_multiqc
set val(name), file("*.M-bias.txt") into ch_bismark_mbias_for_bismark_report, ch_bismark_mbias_for_bismark_summary, ch_bismark_mbias_for_multiqc
file '*.{png,gz}'
set val(name), file("*.CX_report.txt") into ch_bismark_to_cgmap_OG
script:
comprehensive = params.comprehensive ? '--comprehensive --merge_non_CpG' : ''
cytosine_report = params.cytosine_report ? "--cytosine_report --genome_folder ${index} " : ''
meth_cutoff = params.meth_cutoff ? "--cutoff ${params.meth_cutoff}" : ''
multicore = ''
if( task.cpus ){
// Numbers based on Bismark docs
ccore = ((task.cpus as int) / 3) as int
if( ccore > 1 ){
multicore = "--multicore $ccore"
}
}
buffer = ''
if( task.memory ){
mbuffer = (task.memory as nextflow.util.MemoryUnit) - 2.GB
// only set if we have more than 6GB available
if( mbuffer.compareTo(4.GB) == 1 ){
buffer = "--buffer_size ${mbuffer.toGiga()}G"
}
}
if(params.single_end) {
"""
bismark_methylation_extractor $comprehensive $meth_cutoff \\
$multicore $buffer $cytosine_report \\
--bedGraph \\
--counts \\
--gzip \\
-s \\
--report \\
$bam
"""
} else {
"""
bismark_methylation_extractor $comprehensive $meth_cutoff \\
$multicore $buffer \\
--CX_context
--cytosine_report \\
--ignore_r2 2 \\
--ignore_3prime_r2 2 \\
--bedGraph \\
--counts \\
--gzip \\
-p \\
--no_overlap \\
--report \\
$bam
"""
}
}
ch_bismark_align_log_for_bismark_report
.join(ch_bismark_dedup_log_for_bismark_report)
.join(ch_bismark_splitting_report_for_bismark_report)
.join(ch_bismark_mbias_for_bismark_report)
.set{ ch_bismark_logs_for_bismark_report }
/*
* STEP 6 - Bismark Sample Report
*/
process bismark_report {
tag "$name"
publishDir "${params.outdir}/bismark_reports", mode: params.publish_dir_mode
input:
set val(name), file(align_log), file(dedup_log), file(splitting_report), file(mbias) from ch_bismark_logs_for_bismark_report
output:
file '*{html,txt}' into ch_bismark_reports_results_for_multiqc
script:
"""
bismark2report \\
--alignment_report $align_log \\
--dedup_report $dedup_log \\
--splitting_report $splitting_report \\
--mbias_report $mbias
"""
}
/*
* STEP 7 - Bismark Summary Report
*/
process bismark_summary {
publishDir "${params.outdir}/bismark_summary", mode: params.publish_dir_mode
input:
file ('*') from ch_bam_for_bismark_summary.collect()
file ('*') from ch_bismark_align_log_for_bismark_summary.collect()
file ('*') from ch_bismark_dedup_log_for_bismark_summary.collect()
file ('*') from ch_bismark_splitting_report_for_bismark_summary.collect()
file ('*') from ch_bismark_mbias_for_bismark_summary.collect()
output:
file '*{html,txt}' into ch_bismark_summary_results_for_multiqc
script:
"""
bismark2summary
"""
}
} // End of bismark processing block
else {
ch_bismark_align_log_for_multiqc = Channel.from(false)
ch_bismark_dedup_log_for_multiqc = Channel.from(false)
ch_bismark_splitting_report_for_multiqc = Channel.from(false)
ch_bismark_mbias_for_multiqc = Channel.from(false)
ch_bismark_reports_results_for_multiqc = Channel.from(false)
ch_bismark_summary_results_for_multiqc = Channel.from(false)
}
/*
* Process with bwa-mem and assorted tools
*/
if( params.aligner == 'bwameth' ){
process bwamem_align {
tag "$name"
publishDir "${params.outdir}/bwa-mem_alignments", mode: params.publish_dir_mode,
saveAs: {filename ->
if( !params.save_align_intermeds && filename == "where_are_my_files.txt" ) filename
else if( params.save_align_intermeds && filename != "where_are_my_files.txt" ) filename
else null
}
input:
set val(name), file(reads) from ch_trimmed_reads_for_alignment
file bwa_meth_indices from ch_bwa_meth_indices_for_bwamem_align.collect()
file wherearemyfiles from ch_wherearemyfiles_for_bwamem_align.collect()
output:
set val(name), file('*.bam') into ch_bam_for_samtools_sort_index_flagstat, ch_bam_for_preseq, ch_bam_cgmaptools
file "where_are_my_files.txt"
script:
fasta = bwa_meth_indices[0].toString() - '.bwameth' - '.c2t' - '.amb' - '.ann' - '.bwt' - '.pac' - '.sa'
prefix = reads[0].toString() - ~/(_R1)?(_trimmed)?(_val_1)?(\.fq)?(\.fastq)?(\.gz)?$/
"""
bwameth.py \\
--threads ${task.cpus} \\
--reference $fasta \\
$reads | samtools view -bS - > ${prefix}.bam
"""
}
/*
* STEP 4.- samtools flagstat on samples
*/
if ( params.skip_alignment || params.aligner == 'bwameth' )
process samtools_sort_index_flagstat {
tag "$name"
publishDir "${params.outdir}/bam_processing", mode: params.publish_dir_mode,
saveAs: {filename ->
if(filename.indexOf("report.txt") > 0) "logs/$filename"
else if( (!params.save_align_intermeds && !params.skip_deduplication && !params.rrbs).every() && filename == "where_are_my_files.txt") filename
else if( (params.save_align_intermeds || params.skip_deduplication || params.rrbs).any() && filename != "where_are_my_files.txt") filename
else null
}
input:
set val(name), file(bam) from ch_bam_for_samtools_sort_index_flagstat
file wherearemyfiles from ch_wherearemyfiles_for_samtools_sort_index_flagstat.collect()
output:
set val(name), file("${bam.baseName}.sorted.bam") into ch_indep_bam_sorted, ch_bam_sorted_for_markDuplicates
set val(name), file("${bam.baseName}.sorted.bam.bai") into ch_bam_index, ch_indep_bam_index
file "${bam.baseName}_flagstat_report.txt" into ch_flagstat_results_for_multiqc
file "${bam.baseName}_stats_report.txt" into ch_samtools_stats_results_for_multiqc
file "where_are_my_files.txt"
script:
def avail_mem = task.memory ? ((task.memory.toGiga() - 6) / task.cpus).trunc() : false
def sort_mem = avail_mem && avail_mem > 2 ? "-m ${avail_mem}G" : ''
"""
samtools sort $bam \\
-@ ${task.cpus} $sort_mem \\
-o ${bam.baseName}.sorted.bam
samtools index ${bam.baseName}.sorted.bam
samtools flagstat ${bam.baseName}.sorted.bam > ${bam.baseName}_flagstat_report.txt
samtools stats ${bam.baseName}.sorted.bam > ${bam.baseName}_stats_report.txt
"""
}
/*
* STEP 5 - Mark duplicates
*/
if( params.skip_deduplication || params.rrbs ) {
ch_bam_sorted_for_markDuplicates.into { ch_bam_dedup_for_methyldackel; ch_bam_dedup_for_qualimap; ch_bam_cgmaptools }
ch_bam_index.set { ch_bam_index_for_methyldackel }
ch_markDups_results_for_multiqc = Channel.from(false)
} else {
process markDuplicates {
tag "$name"
publishDir "${params.outdir}/bwa-mem_markDuplicates", mode: params.publish_dir_mode,
saveAs: {filename -> filename.indexOf(".bam") == -1 ? "logs/$filename" : "$filename"}
input:
set val(name), file(bam) from ch_bam_sorted_for_markDuplicates
output:
set val(name), file("${name}.markDups.bam") into ch_bam_dedup_for_methyldackel, ch_bam_dedup_for_qualimap, ch_bam_cgmaptools
set val(name), file("${bam.baseName}.markDups.bam.bai") into ch_bam_index_for_methyldackel //ToDo check if this correctly overrides the original channel
file "${bam.baseName}.markDups_metrics.txt" into ch_markDups_results_for_multiqc
script:
if( !task.memory ){
log.info "[Picard MarkDuplicates] Available memory not known - defaulting to 3GB. Specify process memory requirements to change this."
avail_mem = 3
} else {
avail_mem = task.memory.toGiga()
}
"""
picard -Xmx${avail_mem}g MarkDuplicates \\
INPUT=$bam \\
OUTPUT=${bam.baseName}.markDups.bam \\
METRICS_FILE=${bam.baseName}.markDups_metrics.txt \\
REMOVE_DUPLICATES=false \\
ASSUME_SORTED=true \\
PROGRAM_RECORD_ID='null' \\
VALIDATION_STRINGENCY=LENIENT
samtools index ${bam.baseName}.markDups.bam
"""
}
}
/*
* STEP 6 - extract methylation with MethylDackel
*/
process methyldackel {
tag "$name"
publishDir "${params.outdir}/MethylDackel", mode: params.publish_dir_mode
input:
set val(name),
file(bam),
file(bam_index),
file(fasta),
file(fasta_index) from ch_bam_dedup_for_methyldackel
.join(ch_bam_index_for_methyldackel)
.combine(ch_fasta_for_methyldackel)
.combine(ch_fasta_index_for_methyldackel)
output:
file "${bam.baseName}*" into ch_methyldackel_results_for_multiqc
script:
all_contexts = params.comprehensive ? '--CHG --CHH' : ''
min_depth = params.min_depth > 0 ? "--minDepth ${params.min_depth}" : ''
ignore_flags = params.ignore_flags ? "--ignoreFlags" : ''
methyl_kit = params.methyl_kit ? "--methylKit" : ''
"""
MethylDackel extract $all_contexts $ignore_flags $methyl_kit $min_depth $fasta $bam
MethylDackel mbias $all_contexts $ignore_flags $fasta $bam ${bam.baseName} --txt > ${bam.baseName}_methyldackel.txt
"""
}
} // end of bwa-meth if block
else {
ch_flagstat_results_for_multiqc = Channel.from(false)
ch_samtools_stats_results_for_multiqc = Channel.from(false)
ch_markDups_results_for_multiqc = Channel.from(false)
ch_methyldackel_results_for_multiqc = Channel.from(false)
}
/*BismarkIndex_for methXtract */
if( params.aligner == 'none' ){
process makeBismarkIndex_2 {
publishDir path: { params.save_reference ? "${params.outdir}/reference_genome" : params.outdir },
saveAs: { params.save_reference ? it : null }, mode: params.publish_dir_mode
input:
file fasta from ch_fasta_bismarkIndex_2
output:
file "BismarkIndex" into ch_bismark_index_for_bismark_methXtract_2
script:
"""
mkdir BismarkIndex
cp $fasta BismarkIndex/
bismark_genome_preparation --bowtie2 BismarkIndex
"""
}
}
/* Deduplicate for BAM file input */
if (params.aligner == 'none') {
process markDuplicates_bam_input {
tag "${name}"
publishDir "${params.outdir}/bam_markDuplicates", mode: params.publish_dir_mode,
saveAs: {filename -> "$filename"}
input:
//set bam from ch_indep_bam_for_processing
set val(name), file(bam) from ch_indep_bam_for_processing
output:
set val(name), file("*.markDups.bam") into ch_bam_resort, ch_bam_dedup_for_qualimap_indep
set val(name), file("*.markDups.bam.bai") into ch_bam_index_indep
file "*.markDups_metrics.txt" into ch_markDups_results_for_multiqc_indep
script:
//name = bam[0].toString() - ~/(.bam)?$/
if( !task.memory ){
log.info "[Picard MarkDuplicates] Available memory not known - defaulting to 3GB. Specify process memory requirements to change this."
avail_mem = 3
} else {
avail_mem = task.memory.toGiga()
}
"""
picard -Xmx${avail_mem}g MarkDuplicates \\
INPUT=$bam \\
OUTPUT=${name}.markDups.bam \\
METRICS_FILE=${name}.markDups_metrics.txt \\
REMOVE_DUPLICATES=false \\
ASSUME_SORTED=true \\
PROGRAM_RECORD_ID='null' \\
VALIDATION_STRINGENCY=LENIENT
samtools index ${name}.markDups.bam
"""
}
}
/*Sort bam file by RG
*/
if (params.aligner == 'none') {
process sort_bam_RG {
tag "${name}"
publishDir "${params.outdir}/bam_sort_RG", mode: params.publish_dir_mode
input:
set val(name), file(bam) from ch_bam_resort
output:
set val(name), file("*.sorted.bam") into ch_bam_meth_call
script:
"""
samtools sort -n -@ 6 -m 3G $bam -o ${name}.sorted.bam
"""