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#!/usr/bin/env nextflow
/*
------------------------------------------------------------------------------------------------------------
nf-core/eager
------------------------------------------------------------------------------------------------------------
EAGER Analysis Pipeline. Started 2018-06-05
#### Homepage / Documentation
https://github.com/nf-core/eager
#### Authors
For a list of authors and contributors, see: https://github.com/nf-core/eager/tree/dev#authors-alphabetical
------------------------------------------------------------------------------------------------------------
*/
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/eager --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)
}
// Validate BAM input isn't set to paired_end
if ( params.bam && !params.single_end ) {
exit 1, "[nf-core/eager] error: bams can only be specified with --single_end. Please check input command."
}
// Validate that skip_collapse is only set to True for paired_end reads!
if (!has_extension(params.input, "tsv") && params.skip_collapse && params.single_end){
exit 1, "[nf-core/eager] error: --skip_collapse can only be set for paired_end samples."
}
// Validate not trying to both skip collapse and skip trim
if ( params.skip_collapse && params.skip_trim ) {
exit 1, "[nf-core/eager error]: you have specified to skip both merging and trimming of paired end samples. Use --skip_adapterremoval instead."
}
// Bedtools validation
if( params.run_bedtools_coverage && !params.anno_file ){
exit 1, "[nf-core/eager] error: you have turned on bedtools coverage, but not specified a BED or GFF file with --anno_file. Please validate your parameters."
}
// Bedtools validation
if( !params.skip_preseq && !( params.preseq_mode == 'c_curve' || params.preseq_mode == 'lc_extrap' ) ) {
exit 1, "[nf-core/eager] error: you are running preseq with a unsupported mode. See documentation for more information. You gave: ${params.preseq_mode}."
}
// BAM filtering validation
if (!params.run_bam_filtering && params.bam_mapping_quality_threshold != 0) {
exit 1, "[nf-core/eager] error: please turn on BAM filtering if you want to perform mapping quality filtering! Provide: --run_bam_filtering."
}
if (params.dedupper == 'dedup' && !params.mergedonly) {
log.warn "[nf-core/eager] Warning: you are using DeDup but without specifying --mergedonly for AdapterRemoval, dedup will likely fail! See documentation for more information."
}
// Genotyping validation
if (params.run_genotyping){
if (params.genotyping_tool == 'pileupcaller' && ( !params.pileupcaller_bedfile || !params.pileupcaller_snpfile ) ) {
exit 1, "[nf-core/eager] error: please check your pileupCaller bed file and snp file parameters. You must supply a bed file and a snp file."
}
if (params.genotyping_tool == 'angsd' && ! ( params.angsd_glformat == 'text' || params.angsd_glformat == 'binary' || params.angsd_glformat == 'binary_three' || params.angsd_glformat == 'beagle' ) ) {
exit 1, "[nf-core/eager] error: please check your ANGSD output format! Options: 'text', 'binary', 'binary_three', 'beagle'. Found parameter: --angsd_glformat '${params.angsd_glformat}'."
}
}
// Consensus sequence generation validation
if (params.run_vcf2genome) {
if (!params.run_genotyping) {
exit 1, "[nf-core/eager] error: consensus sequence generation requires genotyping via UnifiedGenotyper on be turned on with the parameter --run_genotyping and --genotyping_tool 'ug'. Please check your genotyping parameters."
}
if (params.genotyping_tool != 'ug') {
exit 1, "[nf-core/eager] error: consensus sequence generation requires genotyping via UnifiedGenotyper on be turned on with the parameter --run_genotyping and --genotyping_tool 'ug'. Found parameter: --genotyping_tool '${params.genotyping_tool}'."
}
}
// MultiVCFAnalyzer validation
if (params.run_multivcfanalyzer) {
if (!params.run_genotyping) {
exit 1, "[nf-core/eager] error: MultiVCFAnalyzer requires genotyping to be turned on with the parameter --run_genotyping. Please check your genotyping parameters."
}
if (params.genotyping_tool != "ug") {
exit 1, "[nf-core/eager] error: MultiVCFAnalyzer only accepts VCF files from GATK UnifiedGenotyper. Found parameter: --genotyping_tool '${params.genotyping_tool}'."
}
if (params.gatk_ploidy != 2) {
exit 1, "[nf-core/eager] error: MultiVCFAnalyzer only accepts VCF files generated with a GATK ploidy set to 2. Found parameter: --gatk_ploidy ${params.gatk_ploidy}."
}
if (params.additional_vcf_files) {
ch_extravcfs_for_multivcfanalyzer = Channel.fromPath(params.additional_vcf_files, checkIfExists: true)
}
}
if (params.run_metagenomic_screening) {
if ( !params.run_bam_filtering ) {
exit 1, "[nf-core/eager] error: metagenomic classification can only run on unmapped reads. Please supply --run_bam_filtering --bam_unmapped_type 'fastq'."
}
if ( params.bam_unmapped_type != "fastq" ) {
exit 1, "[nf-core/eager] error: metagenomic classification can only run on unmapped reads. Please supply --bam_unmapped_type 'fastq'. Supplied: --bam_unmapped_type '${params.bam_unmapped_type}'."
}
if (!params.database) {
exit 1, "[nf-core/eager] error: metagenomic classification requires a path to a database directory. Please specify one with --database '/path/to/database/'."
}
if (params.metagenomic_tool == 'malt' && params.malt_min_support_mode == 'percent' && params.metagenomic_min_support_reads != 1) {
exit 1, "[nf-core/eager] error: incompatible MALT min support configuration. Percent can only be used with --malt_min_support_percent. You modified: --metagenomic_min_support_reads."
}
if (params.metagenomic_tool == 'malt' && params.malt_min_support_mode == 'reads' && params.malt_min_support_percent != 0.01) {
exit 1, "[nf-core/eager] error: incompatible MALT min support configuration. Reads can only be used with --malt_min_supportreads. You modified: --malt_min_support_percent."
}
if (!params.metagenomic_min_support_reads.toString().isInteger()){
exit 1, "[nf-core/eager] error: incompatible min_support_reads configuration. min_support_reads can only be used with integers. --metagenomic_min_support_reads Found parameter: ${params.metagenomic_min_support_reads}."
}
}
// MaltExtract validation
if (params.run_maltextract) {
if (params.run_metagenomic_screening && params.metagenomic_tool != 'malt') {
exit 1, "[nf-core/eager] error: MaltExtract can only accept MALT output. Please supply --metagenomic_tool 'malt'. Found parameter: --metagenomic_tool '${params.metagenomic_tool}'"
}
if (params.run_metagenomic_screening && params.metagenomic_tool != 'malt') {
exit 1, "[nf-core/eager] error: MaltExtract can only accept MALT output. Please supply --metagenomic_tool 'malt'. Found parameter: --metagenomic_tool '${params.metagenomic_tool}'"
}
if (!params.maltextract_taxon_list) {
exit 1, "[nf-core/eager] error: MaltExtract requires a taxon list specifying the target taxa of interest. Specify the file with --params.maltextract_taxon_list."
}
}
/////////////////////////////////////////////////////////
/* -- VALIDATE INPUT FILES -- */
/////////////////////////////////////////////////////////
// Set up channels for annotation file
if (!params.run_bedtools_coverage){
ch_anno_for_bedtools = Channel.empty()
} else {
ch_anno_for_bedtools = Channel.fromPath(params.anno_file, checkIfExists: true)
.ifEmpty { exit 1, "[nf-core/eager] error: bedtools annotation file not found. Supplied parameter: --anno_file ${params.anno_file}."}
}
if (params.fasta) {
file(params.fasta, checkIfExists: true)
lastPath = params.fasta.lastIndexOf(File.separator)
lastExt = params.fasta.lastIndexOf(".")
fasta_base = params.fasta.substring(lastPath+1)
index_base = params.fasta.substring(lastPath+1,lastExt)
if (params.fasta.endsWith('.gz')) {
fasta_base = params.fasta.substring(lastPath+1,lastExt)
index_base = fasta_base.substring(0,fasta_base.lastIndexOf("."))
}
} else {
exit 1, "[nf-core/eager] error: please specify --fasta with the path to your reference"
}
// Validate reference inputs
if("${params.fasta}".endsWith(".gz")){
process unzip_reference{
tag "${zipped_fasta}"
input:
path zipped_fasta from file(params.fasta) // path doesn't like it if a string of an object is not prefaced with a root dir (/), so use file() to resolve string before parsing to `path`
output:
path "$unzip" into ch_fasta into ch_fasta_for_bwaindex,ch_fasta_for_bt2index,ch_fasta_for_faidx,ch_fasta_for_seqdict,ch_fasta_for_circulargenerator,ch_fasta_for_circularmapper,ch_fasta_for_damageprofiler,ch_fasta_for_qualimap,ch_unmasked_fasta_for_masking,ch_unmasked_fasta_for_pmdtools,ch_fasta_for_genotyping_ug,ch_fasta_for_genotyping_hc,ch_fasta_for_genotyping_freebayes,ch_fasta_for_genotyping_pileupcaller,ch_fasta_for_vcf2genome,ch_fasta_for_multivcfanalyzer,ch_fasta_for_genotyping_angsd,ch_fasta_for_damagerescaling,ch_fasta_for_bcftools_stats
script:
unzip = zipped_fasta.toString() - '.gz'
"""
pigz -f -d -p ${task.cpus} $zipped_fasta
"""
}
} else {
fasta_for_indexing = Channel
.fromPath("${params.fasta}", checkIfExists: true)
.into{ ch_fasta_for_bwaindex; ch_fasta_for_bt2index; ch_fasta_for_faidx; ch_fasta_for_seqdict; ch_fasta_for_circulargenerator; ch_fasta_for_circularmapper; ch_fasta_for_damageprofiler; ch_fasta_for_qualimap; ch_unmasked_fasta_for_masking; ch_unmasked_fasta_for_pmdtools; ch_fasta_for_genotyping_ug; ch_fasta__for_genotyping_hc; ch_fasta_for_genotyping_hc; ch_fasta_for_genotyping_freebayes; ch_fasta_for_genotyping_pileupcaller; ch_fasta_for_vcf2genome; ch_fasta_for_multivcfanalyzer; ch_fasta_for_genotyping_angsd; ch_fasta_for_damagerescaling; ch_fasta_for_bcftools_stats }
}
// Check that fasta index file path ends in '.fai'
if (params.fasta_index && !params.fasta_index.endsWith(".fai")) {
exit 1, "The specified fasta index file (${params.fasta_index}) is not valid. Fasta index files should end in '.fai'."
}
// Check if genome exists in the config file. params.genomes is from igenomes.conf, params.genome specified by user
if (params.genomes && params.genome && !params.genomes.containsKey(params.genome)) {
exit 1, "The provided genome '${params.genome}' is not available in the iGenomes file. Currently the available genomes are ${params.genomes.keySet().join(', ')}"
}
// Index files provided? Then check whether they are correct and complete
if( params.bwa_index && (params.mapper == 'bwaaln' | params.mapper == 'bwamem' | params.mapper == 'circularmapper')){
Channel
.fromPath(params.bwa_index, checkIfExists: true)
.ifEmpty { exit 1, "[nf-core/eager] error: bwa indices not found in: ${index_base}." }
.into {bwa_index; bwa_index_bwamem}
bt2_index = Channel.empty()
}
if( params.bt2_index && params.mapper == 'bowtie2' ){
lastPath = params.bt2_index.lastIndexOf(File.separator)
bt2_dir = params.bt2_index.substring(0,lastPath+1)
bt2_base = params.bt2_index.substring(lastPath+1)
Channel
.fromPath(params.bt2_index, checkIfExists: true)
.ifEmpty { exit 1, "[nf-core/eager] error: bowtie2 indices not found in: ${bt2_dir}." }
.into {bt2_index; bt2_index_bwamem}
bwa_index = Channel.empty()
bwa_index_bwamem = Channel.empty()
}
// Adapter removal adapter-list setup
if ( !params.clip_adapters_list ) {
Channel
.fromPath("$projectDir/assets/nf-core_eager_dummy2.txt", checkIfExists: true)
.ifEmpty { exit 1, "[nf-core/eager] error: adapters list file not found. Please check input. Supplied: --clip_adapters_list '${params.clip_adapters_list}'." }
.collect()
.set {ch_adapterlist}
} else {
Channel
.fromPath("${params.clip_adapters_list}", checkIfExists: true)
.ifEmpty { exit 1, "[nf-core/eager] error: adapters list file not found. Please check input. Supplied: --clip_adapters_list '${params.clip_adapters_list}'." }
.collect()
.set {ch_adapterlist}
}
if ( params.snpcapture_bed ) {
ch_snpcapture_bed = Channel.fromPath(params.snpcapture_bed, checkIfExists: true).collect()
} else {
ch_snpcapture_bed = Channel.fromPath("$projectDir/assets/nf-core_eager_dummy.txt").collect()
}
// Set up channel with pmdtools reference mask bedfile
if (!params.pmdtools_reference_mask) {
ch_bedfile_for_reference_masking = Channel.fromPath("$projectDir/assets/nf-core_eager_dummy.txt").collect()
} else {
ch_bedfile_for_reference_masking = Channel.fromPath(params.pmdtools_reference_mask, checkIfExists: true).collect()
}
// SexDetermination channel set up and bedfile validation
if (!params.sexdeterrmine_bedfile) {
ch_bed_for_sexdeterrmine = Channel.fromPath("$projectDir/assets/nf-core_eager_dummy.txt").collect()
} else {
ch_bed_for_sexdeterrmine = Channel.fromPath(params.sexdeterrmine_bedfile, checkIfExists: true).collect()
}
// pileupCaller channel generation and input checks for 'random sampling' genotyping
if (!params.pileupcaller_bedfile) {
ch_bed_for_pileupcaller = Channel.fromPath("$projectDir/assets/nf-core_eager_dummy.txt").collect()
} else {
ch_bed_for_pileupcaller = Channel.fromPath(params.pileupcaller_bedfile, checkIfExists: true).collect()
}
if (!params.pileupcaller_snpfile) {
ch_snp_for_pileupcaller = Channel.fromPath("$projectDir/assets/nf-core_eager_dummy2.txt").collect()
} else {
ch_snp_for_pileupcaller = Channel.fromPath(params.pileupcaller_snpfile, checkIfExists: true).collect()
}
// Create input channel for MALT database directory, checking directory exists
if ( !params.database ) {
ch_db_for_malt = Channel.empty()
} else {
ch_db_for_malt = Channel.fromPath(params.database, checkIfExists: true)
}
// Create input channel for MaltExtract taxon list, to allow downloading of taxon list, checking file exists.
if ( !params.maltextract_taxon_list ) {
ch_taxonlist_for_maltextract = Channel.empty()
} else {
ch_taxonlist_for_maltextract = Channel.fromPath(params.maltextract_taxon_list, checkIfExists: true)
}
// Create input channel for MaltExtract NCBI files, checking files exists.
if ( !params.maltextract_ncbifiles ) {
ch_ncbifiles_for_maltextract = Channel.empty()
} else {
ch_ncbifiles_for_maltextract = Channel.fromPath(params.maltextract_ncbifiles, checkIfExists: true)
}
////////////////////////////////////////////////////
/* -- Collect configuration parameters -- */
////////////////////////////////////////////////////
// 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 ${params.genomes.keySet().join(', ')}"
}
// Check AWS batch settings
if (workflow.profile.contains('awsbatch')) {
// AWSBatch sanity checking
if (!params.awsqueue || !params.awsregion) exit 1, 'Specify correct --awsqueue and --awsregion parameters on AWSBatch!'
// Check outdir paths to be S3 buckets if running on AWSBatch
// related: https://github.com/nextflow-io/nextflow/issues/813
if (!params.outdir.startsWith('s3:')) exit 1, 'Outdir not on S3 - specify S3 Bucket to run on AWSBatch!'
// Prevent trace files to be stored on S3 since S3 does not support rolling files.
if (params.tracedir.startsWith('s3:')) exit 1, 'Specify a local tracedir or run without trace! S3 cannot be used for tracefiles.'
}
ch_multiqc_config = file("$projectDir/assets/multiqc_config.yaml", checkIfExists: true)
ch_multiqc_custom_config = params.multiqc_config ? Channel.fromPath(params.multiqc_config, checkIfExists: true) : Channel.empty()
ch_eager_logo = file("$projectDir/docs/images/nf-core_eager_logo_outline_drop.png")
ch_output_docs = file("$projectDir/docs/output.md", checkIfExists: true)
ch_output_docs_images = file("$projectDir/docs/images/", checkIfExists: true)
where_are_my_files = file("$projectDir/assets/where_are_my_files.txt")
///////////////////////////////////////////////////
/* -- INPUT FILE LOADING AND VALIDATING -- */
///////////////////////////////////////////////////
// check if we have valid --reads or --input
if (!params.input) {
exit 1, "[nf-core/eager] error: --input was not supplied! Please check '--help' or documentation under 'running the pipeline' for details"
}
// Read in files properly from TSV file
tsv_path = null
if (params.input && (has_extension(params.input, "tsv"))) tsv_path = params.input
ch_input_sample = Channel.empty()
if (tsv_path) {
tsv_file = file(tsv_path)
if (tsv_file instanceof List) exit 1, "[nf-core/eager] error: can only accept one TSV file per run."
if (!tsv_file.exists()) exit 1, "[nf-core/eager] error: input TSV file could not be found. Does the file exist and is it in the right place? You gave the path: ${params.input}"
ch_input_sample = extract_data(tsv_path)
} else if (params.input && !has_extension(params.input, "tsv")) {
log.info ""
log.info "No TSV file provided - creating TSV from supplied directory."
log.info "Reading path(s): ${params.input}\n"
inputSample = retrieve_input_paths(params.input, params.colour_chemistry, params.single_end, params.single_stranded, params.udg_type, params.bam)
ch_input_sample = inputSample
} else exit 1, "[nf-core/eager] error: --input file(s) not correctly not supplied or improperly defined, see '--help' flag and documentation under 'running the pipeline' for details."
ch_input_sample
.into { ch_input_sample_downstream; ch_input_sample_check }
///////////////////////////////////////////////////
/* -- INPUT CHANNEL CREATION -- */
///////////////////////////////////////////////////
// Check we don't have any duplicate file names
ch_input_sample_check
.map {
it ->
def r1 = file(it[8]).getName()
def r2 = file(it[9]).getName()
def bam = file(it[10]).getName()
[r1, r2, bam]
}
.collect()
.map{
file ->
filenames = file
filenames -= 'NA'
if( filenames.size() != filenames.unique().size() )
exit 1, "[nf-core/eager] error: You have duplicate input FASTQ and/or BAM file names! All files must have unique names, different directories are not sufficent. Please check your input."
}
// Drop samples with R1/R2 to fastQ channel, BAM samples to other channel
ch_branched_input = ch_input_sample_downstream.branch{
fastq: it[8] != 'NA' //These are all fastqs
bam: it[10] != 'NA' //These are all BAMs
}
//Removing BAM/BAI in case of a FASTQ input
ch_fastq_channel = ch_branched_input.fastq.map {
samplename, libraryid, lane, colour, seqtype, organism, strandedness, udg, r1, r2, bam ->
[samplename, libraryid, lane, colour, seqtype, organism, strandedness, udg, r1, r2]
}
//Removing R1/R2 in case of BAM input
ch_bam_channel = ch_branched_input.bam.map {
samplename, libraryid, lane, colour, seqtype, organism, strandedness, udg, r1, r2, bam ->
[samplename, libraryid, lane, colour, seqtype, organism, strandedness, udg, bam]
}
// Prepare starting channels, here we go
ch_input_for_convertbam = Channel.empty()
ch_bam_channel
.into { ch_input_for_convertbam; ch_input_for_indexbam; }
// Also need to send raw files for lane merging, if we want to host removed fastq
ch_fastq_channel
.into { ch_input_for_skipconvertbam; ch_input_for_lanemerge_hostremovalfastq }
////////////////////////////////////////////////////
/* -- 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['Input'] = params.input
summary['Fasta Ref'] = params.fasta
summary['Max Resources'] = "$params.max_memory memory, $params.max_cpus cpus, $params.max_time time per job"
if (workflow.containerEngine) summary['Container'] = "$workflow.containerEngine - $workflow.container"
summary['Output dir'] = params.outdir
summary['Launch dir'] = workflow.launchDir
summary['Working dir'] = workflow.workDir
summary['Script dir'] = workflow.projectDir
summary['User'] = workflow.userName
if (workflow.profile.contains('awsbatch')) {
summary['AWS Region'] = params.awsregion
summary['AWS Queue'] = params.awsqueue
summary['AWS CLI'] = params.awscli
}
summary['Config Profile'] = workflow.profile
if (params.config_profile_description) summary['Config Profile Description'] = params.config_profile_description
if (params.config_profile_contact) summary['Config Profile Contact'] = params.config_profile_contact
if (params.config_profile_url) summary['Config Profile URL'] = params.config_profile_url
summary['Config Files'] = workflow.configFiles.join(', ')
if (params.email || params.email_on_fail) {
summary['E-mail Address'] = params.email
summary['E-mail on failure'] = params.email_on_fail
summary['MultiQC maxsize'] = params.max_multiqc_email_size
}
Channel.from(summary.collect{ [it.key, it.value] })
.map { k,v -> "<dt>$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-eager-summary'
description: " - this information is collected when the pipeline is started."
section_name: 'nf-core/eager Workflow Summary'
section_href: 'https://github.com/nf-core/eager'
plot_type: 'html'
data: |
<dl class=\"dl-horizontal\">
$x
</dl>
""".stripIndent() }
.set { ch_workflow_summary }
// Check the hostnames against configured profiles
checkHostname()
log.info "Schaffa, Schaffa, Genome Baua!"
///////////////////////////////////////////////////
/* -- REFERENCE FASTA INDEXING -- */
///////////////////////////////////////////////////
// BWA Index
if( !params.bwa_index && params.fasta && (params.mapper == 'bwaaln' || params.mapper == 'bwamem' || params.mapper == 'circularmapper')){
process makeBWAIndex {
label 'sc_medium'
tag "${fasta}"
publishDir path: "${params.outdir}/reference_genome/bwa_index", mode: params.publish_dir_mode, saveAs: { filename ->
if (params.save_reference) filename
else if(!params.save_reference && filename == "where_are_my_files.txt") filename
else null
}
input:
path fasta from ch_fasta_for_bwaindex
path where_are_my_files
output:
path "BWAIndex" into (bwa_index, bwa_index_bwamem)
path "where_are_my_files.txt"
script:
"""
bwa index $fasta
mkdir BWAIndex && mv ${fasta}* BWAIndex
"""
}
bt2_index = Channel.empty()
}
// bowtie2 Index
if( !params.bt2_index && params.fasta && params.mapper == "bowtie2"){
process makeBT2Index {
label 'mc_medium'
tag "${fasta}"
publishDir path: "${params.outdir}/reference_genome/bt2_index", mode: params.publish_dir_mode, saveAs: { filename ->
if (params.save_reference) filename
else if(!params.save_reference && filename == "where_are_my_files.txt") filename
else null
}
input:
path fasta from ch_fasta_for_bt2index
path where_are_my_files
output:
path "BT2Index" into (bt2_index)
path "where_are_my_files.txt"
script:
"""
bowtie2-build --threads ${task.cpus} $fasta $fasta
mkdir BT2Index && mv ${fasta}* BT2Index
"""
}
bwa_index = Channel.empty()
bwa_index_bwamem = Channel.empty()
}
// FASTA Index (FAI)
if (params.fasta_index) {
Channel
.fromPath( params.fasta_index )
.set { ch_fai_for_skipfastaindexing }
} else {
Channel
.empty()
.set { ch_fai_for_skipfastaindexing }
}
process makeFastaIndex {
label 'sc_small'
tag "${fasta}"
publishDir path: "${params.outdir}/reference_genome/fasta_index", mode: params.publish_dir_mode, saveAs: { filename ->
if (params.save_reference) filename
else if(!params.save_reference && filename == "where_are_my_files.txt") filename
else null
}
when: !params.fasta_index && params.fasta && ( params.mapper == 'bwaaln' || params.mapper == 'bwamem' || params.mapper == 'circularmapper')
input:
path fasta from ch_fasta_for_faidx
path where_are_my_files
output:
path "*.fai" into ch_fasta_faidx_index
path "where_are_my_files.txt"
script:
"""
samtools faidx $fasta
"""
}
ch_fai_for_skipfastaindexing.mix(ch_fasta_faidx_index)
.into { ch_fai_for_ug; ch_fai_for_hc; ch_fai_for_freebayes; ch_fai_for_pileupcaller; ch_fai_for_angsd }
// Stage dict index file if supplied, else load it into the channel
if (params.seq_dict) {
Channel
.fromPath( params.seq_dict )
.set { ch_dict_for_skipdict }
} else {
Channel
.empty()
.set { ch_dict_for_skipdict }
}
process makeSeqDict {
label 'sc_medium'
tag "${fasta}"
publishDir path: "${params.outdir}/reference_genome/seq_dict", mode: params.publish_dir_mode, saveAs: { filename ->
if (params.save_reference) filename
else if(!params.save_reference && filename == "where_are_my_files.txt") filename
else null
}
when: !params.seq_dict && params.fasta
input:
path fasta from ch_fasta_for_seqdict
path where_are_my_files
output:
path "*.dict" into ch_seq_dict
path "where_are_my_files.txt"
script:
"""
picard -Xmx${task.memory.toMega()}M CreateSequenceDictionary R=$fasta O="${fasta.baseName}.dict"
"""
}
ch_dict_for_skipdict.mix(ch_seq_dict)
.into { ch_dict_for_ug; ch_dict_for_hc; ch_dict_for_freebayes; ch_dict_for_pileupcaller; ch_dict_for_angsd }
//////////////////////////////////////////////////
/* -- BAM INPUT PREPROCESSING -- */
//////////////////////////////////////////////////
// Convert to FASTQ if re-mapping is requested
process convertBam {
label 'mc_small'
tag "$libraryid"
when:
params.run_convertinputbam
input:
tuple samplename, libraryid, lane, colour, seqtype, organism, strandedness, udg, path(bam) from ch_input_for_convertbam
output:
tuple samplename, libraryid, lane, colour, seqtype, organism, strandedness, udg, path("*fastq.gz"), val('NA') into ch_output_from_convertbam
script:
base = "${bam.baseName}"
"""
samtools fastq -t ${bam} | pigz -p ${task.cpus} > ${base}.converted.fastq.gz
"""
}
// If not converted to FASTQ generate pipeline compatible BAM index file (i.e. with correct samtools version)
process indexinputbam {
label 'sc_small'
tag "$libraryid"
when:
bam != 'NA' && !params.run_convertinputbam
input:
tuple samplename, libraryid, lane, colour, seqtype, organism, strandedness, udg, path(bam) from ch_input_for_indexbam
output:
tuple samplename, libraryid, lane, seqtype, organism, strandedness, udg, path(bam), file("*.{bai,csi}") into ch_indexbam_for_filtering
script:
def size = params.large_ref ? '-c' : ''
"""
samtools index ${bam} ${size}
"""
}
// convertbam bypass
ch_input_for_skipconvertbam.mix(ch_output_from_convertbam)
.into { ch_convertbam_for_fastp; ch_convertbam_for_fastqc }
//////////////////////////////////////////////////
/* -- SEQUENCING QC AND FASTQ PREPROCESSING -- */
//////////////////////////////////////////////////
// Raw sequencing QC - allow user evaluate if sequencing any good?
process fastqc {
label 'mc_small'
tag "${libraryid}_L${lane}"
publishDir "${params.outdir}/fastqc/input_fastq", mode: params.publish_dir_mode,
saveAs: { filename ->
filename.indexOf(".zip") > 0 ? "zips/$filename" : "$filename"
}
input:
tuple samplename, libraryid, lane, colour, seqtype, organism, strandedness, udg, file(r1), file(r2) from ch_convertbam_for_fastqc
output:
path "*_fastqc.{zip,html}" into ch_prefastqc_for_multiqc
when:
!params.skip_fastqc
script:
if ( seqtype == 'PE' ) {
"""
fastqc -t ${task.cpus} -q $r1 $r2
rename 's/_fastqc\\.zip\$/_raw_fastqc.zip/' *_fastqc.zip
rename 's/_fastqc\\.html\$/_raw_fastqc.html/' *_fastqc.html
"""
} else {
"""
fastqc -q $r1
rename 's/_fastqc\\.zip\$/_raw_fastqc.zip/' *_fastqc.zip
rename 's/_fastqc\\.html\$/_raw_fastqc.html/' *_fastqc.html
"""
}
}
// Poly-G clipping for 2-colour chemistry sequencers, to reduce erroenous mapping of sequencing artefacts
if (params.complexity_filter_poly_g) {
ch_input_for_fastp = ch_convertbam_for_fastp.branch{
twocol: it[3] == '2' // Nextseq/Novaseq data with possible sequencing artefact
fourcol: it[3] == '4' // HiSeq/MiSeq data where polyGs would be true
}
} else {
ch_input_for_fastp = ch_convertbam_for_fastp.branch{
twocol: it[3] == "dummy" // seq/Novaseq data with possible sequencing artefact
fourcol: it[3] == '4' || it[3] == '2' // HiSeq/MiSeq data where polyGs would be true
}
}
process fastp {
label 'mc_small'
tag "${libraryid}_L${lane}"
publishDir "${params.outdir}/FastP", mode: params.publish_dir_mode
when:
params.complexity_filter_poly_g
input:
tuple samplename, libraryid, lane, colour, seqtype, organism, strandedness, udg, file(r1), file(r2) from ch_input_for_fastp.twocol
output:
tuple samplename, libraryid, lane, colour, seqtype, organism, strandedness, udg, path("*.pG.fq.gz") into ch_output_from_fastp
path("*.json") into ch_fastp_for_multiqc
script:
if( seqtype == 'SE' ){
"""
fastp --in1 ${r1} --out1 "${r1.baseName}.pG.fq.gz" -A -g --poly_g_min_len "${params.complexity_filter_poly_g_min}" -Q -L -w ${task.cpus} --json "${r1.baseName}"_L${lane}_fastp.json
"""
} else {
"""
fastp --in1 ${r1} --in2 ${r2} --out1 "${r1.baseName}.pG.fq.gz" --out2 "${r2.baseName}.pG.fq.gz" -A -g --poly_g_min_len "${params.complexity_filter_poly_g_min}" -Q -L -w ${task.cpus} --json "${libraryid}"_L${lane}_polyg_fastp.json
"""
}
}
// Colour column only useful for fastp, so dropping now to reduce complexity downstream
ch_input_for_fastp.fourcol
.map {
def samplename = it[0]
def libraryid = it[1]
def lane = it[2]
def seqtype = it[4]
def organism = it[5]
def strandedness = it[6]
def udg = it[7]
def r1 = it[8]
def r2 = seqtype == "PE" ? it[9] : file("$projectDir/assets/nf-core_eager_dummy.txt")
[ samplename, libraryid, lane, seqtype, organism, strandedness, udg, r1, r2 ]
}
.set { ch_skipfastp_for_merge }
ch_output_from_fastp
.map{
def samplename = it[0]
def libraryid = it[1]
def lane = it[2]
def seqtype = it[4]
def organism = it[5]
def strandedness = it[6]
def udg = it[7]
def r1 = it[8] instanceof ArrayList ? it[8].sort()[0] : it[8]
def r2 = seqtype == "PE" ? it[8].sort()[1] : file("$projectDir/assets/nf-core_eager_dummy.txt")
[ samplename, libraryid, lane, seqtype, organism, strandedness, udg, r1, r2 ]
}
.set{ ch_fastp_for_merge }
ch_skipfastp_for_merge.mix(ch_fastp_for_merge)
.into { ch_fastp_for_adapterremoval; ch_fastp_for_skipadapterremoval }
// Sequencing adapter clipping and optional paired-end merging in preparation for mapping
process adapter_removal {
label 'mc_small'
tag "${libraryid}_L${lane}"
publishDir "${params.outdir}/adapterremoval", mode: params.publish_dir_mode
input:
tuple samplename, libraryid, lane, seqtype, organism, strandedness, udg, file(r1), file(r2) from ch_fastp_for_adapterremoval
path adapterlist from ch_adapterlist.collect().dump(tag: "Adapter list")
output:
tuple samplename, libraryid, lane, seqtype, organism, strandedness, udg, path("output/*{combined.fq,.se.truncated,pair1.truncated}.gz") into ch_output_from_adapterremoval_r1
tuple samplename, libraryid, lane, seqtype, organism, strandedness, udg, path("output/*pair2.truncated.gz") optional true into ch_output_from_adapterremoval_r2
tuple samplename, libraryid, lane, seqtype, organism, strandedness, udg, path("output/*.settings") into ch_adapterremoval_logs
when:
!params.skip_adapterremoval
script:
def base = "${r1.baseName}_L${lane}"
def adapters_to_remove = !params.clip_adapters_list ? "--adapter1 ${params.clip_forward_adaptor} --adapter2 ${params.clip_reverse_adaptor}" : "--adapter-list ${adapterlist}"
//This checks whether we skip trimming and defines a variable respectively
def preserve5p = params.preserve5p ? '--preserve5p' : '' // applies to any AR command - doesn't affect output file combination
if ( seqtype == 'PE' && !params.skip_collapse && !params.skip_trim && !params.mergedonly && !params.preserve5p ) {
"""
mkdir -p output
AdapterRemoval --file1 ${r1} --file2 ${r2} --basename ${base}.pe --gzip --threads ${task.cpus} --qualitymax ${params.qualitymax} --collapse ${preserve5p} --trimns --trimqualities ${adapters_to_remove} --minlength ${params.clip_readlength} --minquality ${params.clip_min_read_quality} --minadapteroverlap ${params.min_adap_overlap}
cat *.collapsed.gz *.collapsed.truncated.gz *.singleton.truncated.gz *.pair1.truncated.gz *.pair2.truncated.gz > output/${base}.pe.combined.tmp.fq.gz
mv *.settings output/
## Add R_ and L_ for unmerged reads for DeDup compatibility
AdapterRemovalFixPrefix -Xmx${task.memory.toGiga()}g output/${base}.pe.combined.tmp.fq.gz | pigz -p ${task.cpus - 1} > output/${base}.pe.combined.fq.gz
"""
//PE mode, collapse and trim, outputting all reads, preserving 5p
} else if (seqtype == 'PE' && !params.skip_collapse && !params.skip_trim && !params.mergedonly && params.preserve5p) {
"""
mkdir -p output
AdapterRemoval --file1 ${r1} --file2 ${r2} --basename ${base}.pe --gzip --threads ${task.cpus} --qualitymax ${params.qualitymax} --collapse ${preserve5p} --trimns --trimqualities ${adapters_to_remove} --minlength ${params.clip_readlength} --minquality ${params.clip_min_read_quality} --minadapteroverlap ${params.min_adap_overlap}
cat *.collapsed.gz *.singleton.truncated.gz *.pair1.truncated.gz *.pair2.truncated.gz > output/${base}.pe.combined.tmp.fq.gz
mv *.settings output/
## Add R_ and L_ for unmerged reads for DeDup compatibility
AdapterRemovalFixPrefix -Xmx${task.memory.toGiga()}g output/${base}.pe.combined.tmp.fq.gz | pigz -p ${task.cpus - 1} > output/${base}.pe.combined.fq.gz
"""
// PE mode, collapse and trim but only output collapsed reads
} else if ( seqtype == 'PE' && !params.skip_collapse && !params.skip_trim && params.mergedonly && !params.preserve5p ) {
"""
mkdir -p output
AdapterRemoval --file1 ${r1} --file2 ${r2} --basename ${base}.pe --gzip --threads ${task.cpus} --qualitymax ${params.qualitymax} --collapse ${preserve5p} --trimns --trimqualities ${adapters_to_remove} --minlength ${params.clip_readlength} --minquality ${params.clip_min_read_quality} --minadapteroverlap ${params.min_adap_overlap}
cat *.collapsed.gz *.collapsed.truncated.gz > output/${base}.pe.combined.tmp.fq.gz
## Add R_ and L_ for unmerged reads for DeDup compatibility
AdapterRemovalFixPrefix -Xmx${task.memory.toGiga()}g output/${base}.pe.combined.tmp.fq.gz | pigz -p ${task.cpus - 1} > output/${base}.pe.combined.fq.gz
mv *.settings output/
"""
// PE mode, collapse and trim but only output collapsed reads, preserving 5p
} else if ( seqtype == 'PE' && !params.skip_collapse && !params.skip_trim && params.mergedonly && params.preserve5p ) {
"""
mkdir -p output
AdapterRemoval --file1 ${r1} --file2 ${r2} --basename ${base}.pe --gzip --threads ${task.cpus} --qualitymax ${params.qualitymax} --collapse ${preserve5p} --trimns --trimqualities ${adapters_to_remove} --minlength ${params.clip_readlength} --minquality ${params.clip_min_read_quality} --minadapteroverlap ${params.min_adap_overlap}
cat *.collapsed.gz > output/${base}.pe.combined.tmp.fq.gz
## Add R_ and L_ for unmerged reads for DeDup compatibility
AdapterRemovalFixPrefix -Xmx${task.memory.toGiga()}g output/${base}.pe.combined.tmp.fq.gz | pigz -p ${task.cpus - 1} > output/${base}.pe.combined.fq.gz
mv *.settings output/
"""
// PE mode, collapsing but skip trim, (output all reads). Note: seems to still generate `truncated` files for some reason, so merging for safety.
// Will still do default AR length filtering I guess
} else if ( seqtype == 'PE' && !params.skip_collapse && params.skip_trim && !params.mergedonly ) {
"""
mkdir -p output
AdapterRemoval --file1 ${r1} --file2 ${r2} --basename ${base}.pe --gzip --threads ${task.cpus} --qualitymax ${params.qualitymax} --collapse ${preserve5p} --adapter1 "" --adapter2 ""
cat *.collapsed.gz *.pair1.truncated.gz *.pair2.truncated.gz > output/${base}.pe.combined.tmp.fq.gz
## Add R_ and L_ for unmerged reads for DeDup compatibility
AdapterRemovalFixPrefix -Xmx${task.memory.toGiga()}g output/${base}.pe.combined.tmp.fq.gz | pigz -p ${task.cpus - 1} > output/${base}.pe.combined.fq.gz
mv *.settings output/
"""
// PE mode, collapsing but skip trim, and only output collapsed reads. Note: seems to still generate `truncated` files for some reason, so merging for safety.
// Will still do default AR length filtering I guess
} else if ( seqtype == 'PE' && !params.skip_collapse && params.skip_trim && params.mergedonly ) {
"""
mkdir -p output
AdapterRemoval --file1 ${r1} --file2 ${r2} --basename ${base}.pe --gzip --threads ${task.cpus} --qualitymax ${params.qualitymax} --collapse ${preserve5p} --adapter1 "" --adapter2 ""
cat *.collapsed.gz > output/${base}.pe.combined.tmp.fq.gz
## Add R_ and L_ for unmerged reads for DeDup compatibility
AdapterRemovalFixPrefix -Xmx${task.memory.toGiga()}g output/${base}.pe.combined.tmp.fq.gz | pigz -p ${task.cpus - 1} > output/${base}.pe.combined.fq.gz
mv *.settings output/
"""
// PE mode, skip collapsing but trim (output all reads, as merging not possible) - activates paired-end mapping!
} else if ( seqtype == 'PE' && params.skip_collapse && !params.skip_trim ) {
"""
mkdir -p output
AdapterRemoval --file1 ${r1} --file2 ${r2} --basename ${base}.pe --gzip --threads ${task.cpus} --qualitymax ${params.qualitymax} ${preserve5p} --trimns --trimqualities ${adapters_to_remove} --minlength ${params.clip_readlength} --minquality ${params.clip_min_read_quality} --minadapteroverlap ${params.min_adap_overlap}
mv ${base}.pe.pair*.truncated.gz *.settings output/
"""
} else if ( seqtype != 'PE' && !params.skip_trim ) {
//SE, collapse not possible, trim reads only
"""
mkdir -p output
AdapterRemoval --file1 ${r1} --basename ${base}.se --gzip --threads ${task.cpus} --qualitymax ${params.qualitymax} ${preserve5p} --trimns --trimqualities ${adapters_to_remove} --minlength ${params.clip_readlength} --minquality ${params.clip_min_read_quality} --minadapteroverlap ${params.min_adap_overlap}
mv *.settings *.se.truncated.gz output/
"""
} else if ( seqtype != 'PE' && params.skip_trim ) {
//SE, collapse not possible, trim reads only
"""
mkdir -p output
AdapterRemoval --file1 ${r1} --basename ${base}.se --gzip --threads ${task.cpus} --qualitymax ${params.qualitymax} ${preserve5p} --adapter1 "" --adapter2 ""
mv *.settings *.se.truncated.gz output/
"""
}
}
// When not collapsing paired-end data, re-merge the R1 and R2 files into single map. Otherwise if SE or collapsed PE, R2 now becomes NA
// Sort to make sure we get consistent R1 and R2 ordered when using `-resume`, even if not needed for FastQC
if ( params.skip_collapse ){
ch_output_from_adapterremoval_r1
.mix(ch_output_from_adapterremoval_r2)
.groupTuple(by: [0,1,2,3,4,5,6])
.map{
it ->
def samplename = it[0]
def libraryid = it[1]
def lane = it[2]
def seqtype = it[3]
def organism = it[4]
def strandedness = it[5]
def udg = it[6]
def r1 = file(it[7].sort()[0])
def r2 = seqtype == "PE" ? file(it[7].sort()[1]) : file("$projectDir/assets/nf-core_eager_dummy.txt")
[ samplename, libraryid, lane, seqtype, organism, strandedness, udg, r1, r2 ]
}
.into { ch_output_from_adapterremoval; ch_adapterremoval_for_postfastqc }
} else {
ch_output_from_adapterremoval_r1
.map{
it ->
def samplename = it[0]
def libraryid = it[1]
def lane = it[2]
def seqtype = it[3]
def organism = it[4]
def strandedness = it[5]
def udg = it[6]
def r1 = file(it[7])
def r2 = file("$projectDir/assets/nf-core_eager_dummy.txt")
[ samplename, libraryid, lane, seqtype, organism, strandedness, udg, r1, r2 ]
}
.into { ch_output_from_adapterremoval; ch_adapterremoval_for_postfastqc }
}
// AdapterRemoval bypass when not running it
if (!params.skip_adapterremoval) {
ch_output_from_adapterremoval.mix(ch_fastp_for_skipadapterremoval)
.dump(tag: "post_ar_adapterremoval_decision_skipar")
.filter { it =~/.*combined.fq.gz|.*truncated.gz/ }
.dump(tag: "ar_bypass")
.into { ch_adapterremoval_for_post_ar_trimming; ch_adapterremoval_for_skip_post_ar_trimming; }
} else {
ch_fastp_for_skipadapterremoval
.dump(tag: "post_ar_adapterremoval_decision_withar")
.into { ch_adapterremoval_for_post_ar_trimming; ch_adapterremoval_for_skip_post_ar_trimming; }
}
// Post AR fastq trimming
process post_ar_fastq_trimming {
label 'mc_small'
tag "${libraryid}"
publishDir "${params.outdir}/post_ar_fastq_trimmed", mode: params.publish_dir_mode
when: params.run_post_ar_trimming
input:
tuple samplename, libraryid, lane, seqtype, organism, strandedness, udg, path(r1), path(r2) from ch_adapterremoval_for_post_ar_trimming
output:
tuple samplename, libraryid, lane, seqtype, organism, strandedness, udg, path("*_R1_postartrimmed.fq.gz") into ch_post_ar_trimming_for_lanemerge_r1
tuple samplename, libraryid, lane, seqtype, organism, strandedness, udg, path("*_R2_postartrimmed.fq.gz") optional true into ch_post_ar_trimming_for_lanemerge_r2
script:
if ( seqtype == 'SE' | (seqtype == 'PE' && !params.skip_collapse) ) {
"""
fastp --in1 ${r1} --trim_front1 ${params.post_ar_trim_front} --trim_tail1 ${params.post_ar_trim_tail} -A -G -Q -L -w ${task.cpus} --out1 "${libraryid}"_L"${lane}"_R1_postartrimmed.fq.gz
"""