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#!/usr/bin/env nextflow
/*
========================================================================================
nf-core/viralrecon
========================================================================================
nf-core/viralrecon Analysis Pipeline.
#### Homepage / Documentation
https://github.com/nf-core/viralrecon
----------------------------------------------------------------------------------------
*/
def helpMessage() {
log.info nfcoreHeader()
log.info"""
Usage:
The typical command for running the pipeline is as follows:
nextflow run nf-core/viralrecon --input samplesheet.csv --genome 'MN908947.3' -profile docker
Mandatory arguments
--input [file] Comma-separated file containing information about the samples in the experiment (see docs/usage.md)
--fasta [file] Path to fasta reference for viral genome. Mandatory when --genome not supplied
--amplicon_bed [file] Path to BED file containing amplicon positions. Mandatory when calling variants with --protocol amplicon
--amplicon_fasta [file] Path to fasta file containing amplicon sequences. Mandatory when performing assembly with --protocol amplicon
-profile [str] Configuration profile to use. Can use multiple (comma separated)
Available: conda, docker, singularity, test, awsbatch, <institute> and more
Generic
--protocol [str] Specifies the type of protocol used for sequencing i.e. 'metagenomic' or 'amplicon' (Default: 'metagenomic')
SRA download
--save_sra_fastq [bool] Save FastQ files created from SRA identifiers in the results directory (Default: false)
--skip_sra [bool] Skip steps involving the download and validation of FastQ files using SRA identifiers (Default: false)
References If not specified in the configuration file or you wish to overwrite any of the references
--genome [str] Name of genome reference key for viral genome (Default: '')
--gff [file] Full path to viral gff annotation file (Default: '')
--save_reference [bool] If generated by the pipeline save the Bowtie2 indices in the results directory (Default: false)
Read trimming
--cut_mean_quality [int] The mean quality requirement option shared by fastp cut_front, cut_tail or cut_sliding options. Range: 1~36 (Default: 30 (Q30))
--qualified_quality_phred [int] The quality value that a base is qualified. Default 30 means phred quality >=Q30 is qualified (Default: 30)
--unqualified_percent_limit [int] Percentage of bases that are allowed to be unqualified (0~100) (Default: 10)
--min_trim_length [int] Reads shorter than this length after trimming will be discarded (Default: 50)
--skip_adapter_trimming [bool] Skip the adapter trimming step with fastp (Default: false)
--skip_amplicon_trimming [bool] Skip the amplicon trimming step with Cutadapt (Default: false)
--save_trimmed [bool] Save the trimmed FastQ files in the results directory (Default: false)
Kraken2
--kraken2_db [file] Full path to Kraken2 database built from host genome (Default: kraken2_human.tar.gz hosted on Zenodo)
--kraken2_db_name [str] Name of host genome for building Kraken2 database (Default: 'human')
--kraken2_use_ftp [bool] Use FTP instead of rsync when building kraken2 databases (Default: false)
--save_kraken2_fastq [bool] Save the host and viral fastq files in the results directory (Default: false)
--skip_kraken2 [bool] Skip Kraken2 process for removing host classified reads (Default: false)
Variant calling
--callers [str] Specify which variant calling algorithms you would like to use (Default: 'varscan2,ivar,bcftools')
--min_mapped_reads [int] Minimum number of mapped reads below which samples are removed from further processing (Default: 1000)
--ivar_trim_noprimer [bool] Unset -e parameter for iVar trim. Reads with primers are included by default (Default: false)
--ivar_trim_min_len [int] Minimum length of read to retain after trimming (Default: 20)
--ivar_trim_min_qual [int] Minimum quality threshold for sliding window to pass (Default: 20)
--ivar_trim_window_width [int] Width of sliding window (Default: 4)
--filter_dups [bool] Remove duplicate reads from alignments as identified by picard MarkDuplicates (Default: false)
--filter_unmapped [bool] Remove unmapped reads from alignments (Default: false)
--mpileup_depth [int] SAMTools mpileup max per-file depth (Default: 0)
--min_base_qual [int] When performing variant calling skip bases with baseQ/BAQ smaller than this number (Default: 20)
--min_coverage [int] When performing variant calling skip positions with an overall read depth smaller than this number (Default: 10)
--min_allele_freq [float] Minimum allele frequency threshold for calling variants (Default: 0.25)
--max_allele_freq [float] Maximum allele frequency threshold for filtering variant calls (Default: 0.75)
--varscan2_strand_filter [bool] Ignore Varscan 2 variants with >90% support on one strand (Default: true)
--amplicon_left_suffix [str] Suffix used in name field of --amplicon_bed to indicate left primer position (Default: '_LEFT')
--amplicon_right_suffix [str] Suffix used in name field of --amplicon_bed to indicate right primer position (Default: '_RIGHT')
--save_align_intermeds [bool] Save the intermediate BAM files from the alignment steps (Default: false)
--save_mpileup [bool] Save MPileup files generated during variant calling (Default: false)
--skip_markduplicates [bool] Skip picard MarkDuplicates step (Default: false)
--skip_picard_metrics [bool] Skip Picard CollectMultipleMetrics and CollectWgsMetrics (Default: false)
--skip_mosdepth [bool] Skip genome-wide and amplicon coverage plot generation from mosdepth output (Default: false)
--skip_snpeff [bool] Skip SnpEff and SnpSift annotation of variants (Default: false)
--skip_variants_quast [bool] Skip generation of QUAST aggregated report for consensus sequences (Default: false)
--skip_variants [bool] Skip variant calling steps in the pipeline (Default: false)
De novo assembly
--assemblers [str] Specify which assembly algorithms you would like to use (Default: 'spades,metaspades,unicycler,minia')
--minia_kmer [int] Kmer size to use when running minia (Default: 31)
--skip_blast [bool] Skip blastn of assemblies relative to reference genome (Default: false)
--skip_abacas [bool] Skip ABACAS process for assembly contiguation (Default: false)
--skip_plasmidid [bool] Skip assembly report generation by PlasmidID (Default: false)
--skip_vg [bool] Skip variant graph creation and variant calling relative to reference (Default: false)
--skip_assembly_quast [bool] Skip generation of QUAST aggregated report for assemblies (Default: false)
--skip_assembly [bool] Skip assembly steps in the pipeline (Default: false)
QC
--skip_fastqc [bool] Skip FastQC (Default: false)
--skip_multiqc [bool] Skip MultiQC (Default: false)
Other options:
--outdir [file] The output directory where the results will be saved
--email [email] Set this parameter to your e-mail address to get a summary e-mail with details of the run sent to you when the workflow exits
--email_on_fail [email] Same as --email, except only send mail if the workflow is not successful
--max_multiqc_email_size [str] Theshold size for MultiQC report to be attached in notification email. If file generated by pipeline exceeds the threshold, it will not be attached (Default: 25MB)
-name [str] Name for the pipeline run. If not specified, Nextflow will automatically generate a random mnemonic
AWSBatch options:
--awsqueue [str] The AWSBatch JobQueue that needs to be set when running on AWSBatch
--awsregion [str] The AWS Region for your AWS Batch job to run on
--awscli [str] Path to the AWS CLI tool
""".stripIndent()
}
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/* -- -- */
/* -- SET UP CONFIGURATION VARIABLES -- */
/* -- -- */
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
// Show help message
if (params.help) {
helpMessage()
exit 0
}
// Has the run name been specified by the user?
// this has the bonus effect of catching both -name and --name
custom_runName = params.name
if (!(workflow.runName ==~ /[a-z]+_[a-z]+/)) {
custom_runName = workflow.runName
}
////////////////////////////////////////////////////
/* -- VALIDATE INPUTS -- */
////////////////////////////////////////////////////
if (params.input) { ch_input = file(params.input, checkIfExists: true) } else { exit 1, "Input samplesheet file not specified!" }
if (params.protocol != 'metagenomic' && params.protocol != 'amplicon') {
exit 1, "Invalid protocol option: ${params.protocol}. Valid options: 'metagenomic' or 'amplicon'!"
}
if (params.protocol == 'amplicon' && !params.skip_assembly && !params.amplicon_fasta) {
exit 1, "To perform de novo assembly in 'amplicon' mode please provide a valid amplicon fasta file!"
}
if (params.amplicon_fasta) { ch_amplicon_fasta = file(params.amplicon_fasta, checkIfExists: true) }
if (params.protocol == 'amplicon' && !params.skip_variants && !params.amplicon_bed) {
exit 1, "To perform variant calling in 'amplicon' mode please provide a valid amplicon BED file!"
}
if (params.amplicon_bed) { ch_amplicon_bed = file(params.amplicon_bed, checkIfExists: true) }
callerList = [ 'varscan2', 'ivar', 'bcftools', 'none']
callers = params.callers ? params.callers.split(',').collect{ it.trim().toLowerCase() } : []
if ((callerList + callers).unique().size() != callerList.size()) {
exit 1, "Invalid variant calller option: ${params.callers}. Valid options: ${callerList.join(', ')}"
}
assemblerList = [ 'spades', 'metaspades', 'unicycler', 'minia', 'none' ]
assemblers = params.assemblers ? params.assemblers.split(',').collect{ it.trim().toLowerCase() } : []
if ((assemblerList + assemblers).unique().size() != assemblerList.size()) {
exit 1, "Invalid assembler option: ${params.assemblers}. Valid options: ${assemblerList.join(', ')}"
}
// Viral reference files
if (params.genomes && params.genome && !params.genomes.containsKey(params.genome)) {
exit 1, "The provided genome '${params.genome}' is not available in the Genome file. Currently the available genomes are ${params.genomes.keySet().join(", ")}"
}
params.fasta = params.genome ? params.genomes[ params.genome ].fasta ?: false : false
params.gff = params.genome ? params.genomes[ params.genome ].gff ?: false : false
if (params.fasta) {
file(params.fasta, checkIfExists: true)
lastPath = params.fasta.lastIndexOf(File.separator)
lastExt = params.fasta.lastIndexOf(".")
fasta_base = params.fasta.substring(lastPath+1)
index_base = params.fasta.substring(lastPath+1,lastExt)
if (params.fasta.endsWith('.gz')) {
fasta_base = params.fasta.substring(lastPath+1,lastExt)
index_base = fasta_base.substring(0,fasta_base.lastIndexOf("."))
}
} else {
exit 1, "Viral genome fasta file not specified!"
}
////////////////////////////////////////////////////
/* -- CONFIG FILES -- */
////////////////////////////////////////////////////
ch_multiqc_config = file("$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)
ch_dummy_file = file("$projectDir/assets/dummy_file.txt", checkIfExists: true)
////////////////////////////////////////////////////
/* -- HEADER FILES -- */
////////////////////////////////////////////////////
ch_blast_outfmt6_header = file("$projectDir/assets/headers/blast_outfmt6_header.txt", checkIfExists: true)
ch_ivar_variants_header_mqc = file("$projectDir/assets/headers/ivar_variants_header_mqc.txt", checkIfExists: true)
////////////////////////////////////////////////////
/* -- AWS -- */
////////////////////////////////////////////////////
// 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."
}
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/* -- -- */
/* -- HEADER LOG INFO -- */
/* -- -- */
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
// Header log info
log.info nfcoreHeader()
def summary = [:]
if (workflow.revision) summary['Pipeline Release'] = workflow.revision
summary['Run Name'] = custom_runName ?: workflow.runName
summary['Samplesheet'] = params.input
summary['Protocol'] = params.protocol
if (params.protocol == 'amplicon') summary['Amplicon Fasta File'] = params.amplicon_fasta
if (params.protocol == 'amplicon') summary['Amplicon BED File'] = params.amplicon_bed
if (params.protocol == 'amplicon') summary['Amplicon Left Suffix'] = params.amplicon_left_suffix
if (params.protocol == 'amplicon') summary['Amplicon Right Suffix'] = params.amplicon_right_suffix
summary['Viral Genome'] = params.genome ?: 'Not supplied'
summary['Viral Fasta File'] = params.fasta
if (params.gff) summary['Viral GFF'] = params.gff
if (params.save_reference) summary['Save Genome Indices'] = 'Yes'
if (params.save_sra_fastq) summary['Save SRA FastQ'] = params.save_sra_fastq
if (params.skip_sra) summary['Skip SRA Download'] = params.skip_sra
if (!params.skip_adapter_trimming) {
if (params.cut_mean_quality) summary['Fastp Mean Qual'] = params.cut_mean_quality
if (params.qualified_quality_phred) summary['Fastp Qual Phred'] = params.qualified_quality_phred
if (params.unqualified_percent_limit) summary['Fastp Unqual % Limit'] = params.unqualified_percent_limit
if (params.min_trim_length) summary['Fastp Min Trim Length'] = params.min_trim_length
} else {
summary['Skip Adapter Trimming'] = 'Yes'
}
if (params.skip_amplicon_trimming) summary['Skip Amplicon Trimming'] = 'Yes'
if (params.save_trimmed) summary['Save Trimmed'] = 'Yes'
if (!params.skip_variants) {
summary['Variant Calling Tools'] = params.callers
summary['Min Mapped Reads'] = params.min_mapped_reads
if (params.ivar_trim_noprimer) summary['iVar Trim Exclude'] = 'Yes'
summary['iVar Trim Min Len'] = params.ivar_trim_min_len
summary['iVar Trim Min Qual'] = params.ivar_trim_min_qual
summary['iVar Trim Window'] = params.ivar_trim_window_width
if (params.filter_dups) summary['Remove Duplicate Reads'] = 'Yes'
if (params.filter_unmapped) summary['Remove Unmapped Reads'] = 'Yes'
summary['Mpileup Depth'] = params.mpileup_depth
summary['Min Base Quality'] = params.min_base_qual
summary['Min Read Depth'] = params.min_coverage
summary['Min Allele Freq'] = params.min_allele_freq
summary['Max Allele Freq'] = params.max_allele_freq
if (params.varscan2_strand_filter) summary['Varscan2 Strand Filter'] = 'Yes'
if (params.save_align_intermeds) summary['Save Align Intermeds'] = 'Yes'
if (params.save_mpileup) summary['Save mpileup'] = 'Yes'
if (params.skip_markduplicates) summary['Skip MarkDuplicates'] = 'Yes'
if (params.skip_picard_metrics) summary['Skip Picard Metrics'] = 'Yes'
if (params.skip_mosdepth) summary['Skip mosdepth'] = 'Yes'
if (params.skip_snpeff) summary['Skip SnpEff'] = 'Yes'
if (params.skip_variants_quast) summary['Skip Variants QUAST'] = 'Yes'
} else {
summary['Skip Variant Calling'] = 'Yes'
}
if (!params.skip_kraken2 && !params.skip_assembly) {
if (params.kraken2_db) summary['Host Kraken2 DB'] = params.kraken2_db
if (params.kraken2_db_name) summary['Host Kraken2 Name'] = params.kraken2_db_name
if (params.kraken2_use_ftp) summary['Kraken2 Use FTP'] = params.kraken2_use_ftp
if (params.save_kraken2_fastq) summary['Save Kraken2 FastQ'] = params.save_kraken2_fastq
} else {
summary['Skip Kraken2'] = 'Yes'
}
if (!params.skip_assembly) {
summary['Assembly Tools'] = params.assemblers
summary['Minia Kmer Size'] = params.minia_kmer
if (params.skip_vg) summary['Skip Variant Graph'] = 'Yes'
if (params.skip_blast) summary['Skip BLAST'] = 'Yes'
if (params.skip_abacas) summary['Skip ABACAS'] = 'Yes'
if (params.skip_plasmidid) summary['Skip PlasmidID'] = 'Yes'
if (params.skip_assembly_quast) summary['Skip Assembly QUAST'] = 'Yes'
} else {
summary['Skip Assembly'] = 'Yes'
}
if (params.skip_fastqc) summary['Skip FastQC'] = 'Yes'
if (params.skip_multiqc) summary['Skip MultiQC'] = 'Yes'
summary['Max Resources'] = "$params.max_memory memory, $params.max_cpus cpus, $params.max_time time per job"
if (workflow.containerEngine) summary['Container'] = "$workflow.containerEngine - $workflow.container"
summary['Output dir'] = params.outdir
summary['Publish dir mode'] = params.publish_dir_mode
summary['Launch dir'] = workflow.launchDir
summary['Working dir'] = workflow.workDir
summary['Script dir'] = workflow.projectDir
summary['User'] = workflow.userName
if (workflow.profile.contains('awsbatch')) {
summary['AWS Region'] = params.awsregion
summary['AWS Queue'] = params.awsqueue
summary['AWS CLI'] = params.awscli
}
summary['Config Profile'] = workflow.profile
if (params.config_profile_description) summary['Config Description'] = params.config_profile_description
if (params.config_profile_contact) summary['Config Contact'] = params.config_profile_contact
if (params.config_profile_url) summary['Config URL'] = params.config_profile_url
if (params.email || params.email_on_fail) {
summary['E-mail Address'] = params.email
summary['E-mail on failure'] = params.email_on_fail
summary['MultiQC maxsize'] = params.max_multiqc_email_size
}
log.info summary.collect { k,v -> "${k.padRight(22)}: $v" }.join("\n")
log.info "-\033[2m--------------------------------------------------\033[0m-"
// Check the hostnames against configured profiles
checkHostname()
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/* -- -- */
/* -- UNZIP/UNTAR REFERENCE FILES -- */
/* -- -- */
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/*
* PREPROCESSING: Uncompress genome fasta file
*/
if (params.fasta.endsWith('.gz')) {
process GUNZIP_FASTA {
label 'error_retry'
if (params.save_reference) {
publishDir "${params.outdir}/genome", mode: params.publish_dir_mode
}
input:
path fasta from params.fasta
output:
path "$unzip" into ch_fasta
script:
unzip = fasta.toString() - '.gz'
"""
pigz -f -d -p $task.cpus $fasta
"""
}
} else {
ch_fasta = file(params.fasta)
}
// Print warning if viral genome fasta has more than one sequence
def count = 0
ch_fasta.withReader { reader ->
while (line = reader.readLine()) {
if (line.contains('>')) {
count++
if (count > 1) {
log.info "[nf-core/viralrecon] WARNING: This pipeline does not support multi-fasta genome files. Please amend the '--fasta' parameter."
break
}
}
}
}
/*
* PREPROCESSING: Uncompress gff annotation file
*/
if (params.gff) {
file(params.gff, checkIfExists: true)
if (params.gff.endsWith('.gz')) {
process GUNZIP_GFF {
label 'error_retry'
if (params.save_reference) {
publishDir "${params.outdir}/genome", mode: params.publish_dir_mode
}
input:
path gff from params.gff
output:
path "$unzip" into ch_gff
script:
unzip = gff.toString() - '.gz'
"""
pigz -f -d -p $task.cpus $gff
"""
}
} else {
ch_gff = file(params.gff)
}
} else {
ch_gff = ch_dummy_file
}
/*
* PREPROCESSING: Uncompress Kraken2 database
*/
if (!params.skip_kraken2 && params.kraken2_db && !params.skip_assembly) {
file(params.kraken2_db, checkIfExists: true)
if (params.kraken2_db.endsWith('.tar.gz')) {
process UNTAR_KRAKEN2_DB {
label 'error_retry'
if (params.save_reference) {
publishDir "${params.outdir}/genome", mode: params.publish_dir_mode
}
input:
path db from params.kraken2_db
output:
path "$untar" into ch_kraken2_db
script:
untar = db.toString() - '.tar.gz'
"""
tar -xvf $db
"""
}
} else {
ch_kraken2_db = file(params.kraken2_db)
}
}
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/* -- -- */
/* -- PARSE DESIGN FILE -- */
/* -- -- */
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/*
* PREPROCESSING: Reformat samplesheet and check validity
*/
process CHECK_SAMPLESHEET {
tag "$samplesheet"
publishDir "${params.outdir}/", mode: params.publish_dir_mode,
saveAs: { filename ->
if (filename.endsWith(".tsv")) "preprocess/sra/$filename"
else "pipeline_info/$filename"
}
input:
path samplesheet from ch_input
output:
path "samplesheet.valid.csv" into ch_samplesheet_reformat
path "sra_run_info.tsv" optional true
script: // These scripts are bundled with the pipeline, in nf-core/viralrecon/bin/
run_sra = !params.skip_sra && !isOffline()
"""
awk -F, '{if(\$1 != "" && \$2 != "") {print \$0}}' $samplesheet > nonsra_id.csv
check_samplesheet.py nonsra_id.csv nonsra.samplesheet.csv
awk -F, '{if(\$1 != "" && \$2 == "" && \$3 == "") {print \$1}}' $samplesheet > sra_id.list
if $run_sra && [ -s sra_id.list ]
then
fetch_sra_runinfo.py sra_id.list sra_run_info.tsv --platform ILLUMINA --library_layout SINGLE,PAIRED
sra_runinfo_to_samplesheet.py sra_run_info.tsv sra.samplesheet.csv
fi
if [ -f nonsra.samplesheet.csv ]
then
head -n 1 nonsra.samplesheet.csv > samplesheet.valid.csv
else
head -n 1 sra.samplesheet.csv > samplesheet.valid.csv
fi
tail -n +2 -q *sra.samplesheet.csv >> samplesheet.valid.csv
"""
}
// Function to get list of [ sample, single_end?, is_sra?, is_ftp?, [ fastq_1, fastq_2 ], [ md5_1, md5_2] ]
def validate_input(LinkedHashMap sample) {
def sample_id = sample.sample_id
def single_end = sample.single_end.toBoolean()
def is_sra = sample.is_sra.toBoolean()
def is_ftp = sample.is_ftp.toBoolean()
def fastq_1 = sample.fastq_1
def fastq_2 = sample.fastq_2
def md5_1 = sample.md5_1
def md5_2 = sample.md5_2
def array = []
if (!is_sra) {
if (single_end) {
array = [ sample_id, single_end, is_sra, is_ftp, [ file(fastq_1, checkIfExists: true) ] ]
} else {
array = [ sample_id, single_end, is_sra, is_ftp, [ file(fastq_1, checkIfExists: true), file(fastq_2, checkIfExists: true) ] ]
}
} else {
array = [ sample_id, single_end, is_sra, is_ftp, [ fastq_1, fastq_2 ], [ md5_1, md5_2 ] ]
}
return array
}
/*
* Create channels for input fastq files
*/
ch_samplesheet_reformat
.splitCsv(header:true, sep:',')
.map { validate_input(it) }
.into { ch_reads_all
ch_reads_sra }
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/* -- -- */
/* -- DOWNLOAD SRA FILES -- */
/* -- -- */
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/*
* STEP 1: Download and check SRA data
*/
if (!params.skip_sra || !isOffline()) {
ch_reads_sra
.filter { it[2] }
.into { ch_reads_sra_ftp
ch_reads_sra_dump }
process SRA_FASTQ_FTP {
tag "$sample"
label 'process_medium'
label 'error_retry'
publishDir "${params.outdir}/preprocess/sra", mode: params.publish_dir_mode,
saveAs: { filename ->
if (filename.endsWith(".md5")) "md5/$filename"
else params.save_sra_fastq ? filename : null
}
when:
is_ftp
input:
tuple val(sample), val(single_end), val(is_sra), val(is_ftp), val(fastq), val(md5) from ch_reads_sra_ftp
output:
tuple val(sample), val(single_end), val(is_sra), val(is_ftp), path("*.fastq.gz") into ch_sra_fastq_ftp
path "*.md5"
script:
if (single_end) {
"""
curl -L ${fastq[0]} -o ${sample}.fastq.gz
echo "${md5[0]} ${sample}.fastq.gz" > ${sample}.fastq.gz.md5
md5sum -c ${sample}.fastq.gz.md5
"""
} else {
"""
curl -L ${fastq[0]} -o ${sample}_1.fastq.gz
echo "${md5[0]} ${sample}_1.fastq.gz" > ${sample}_1.fastq.gz.md5
md5sum -c ${sample}_1.fastq.gz.md5
curl -L ${fastq[1]} -o ${sample}_2.fastq.gz
echo "${md5[1]} ${sample}_2.fastq.gz" > ${sample}_2.fastq.gz.md5
md5sum -c ${sample}_2.fastq.gz.md5
"""
}
}
process SRA_FASTQ_DUMP {
tag "$sample"
label 'process_medium'
label 'error_retry'
publishDir "${params.outdir}/preprocess/sra", mode: params.publish_dir_mode,
saveAs: { filename ->
if (filename.endsWith(".log")) "log/$filename"
else params.save_sra_fastq ? filename : null
}
when:
!is_ftp
input:
tuple val(sample), val(single_end), val(is_sra), val(is_ftp) from ch_reads_sra_dump.map { it[0..3] }
output:
tuple val(sample), val(single_end), val(is_sra), val(is_ftp), path("*.fastq.gz") into ch_sra_fastq_dump
path "*.log"
script:
prefix = "${sample.split('_')[0..-2].join('_')}"
pe = single_end ? "" : "--readids --split-e"
rm_orphan = single_end ? "" : "[ -f ${prefix}.fastq.gz ] && rm ${prefix}.fastq.gz"
"""
parallel-fastq-dump \\
--sra-id $prefix \\
--threads $task.cpus \\
--outdir ./ \\
--tmpdir ./ \\
--gzip \\
$pe \\
> ${prefix}.fastq_dump.log
$rm_orphan
"""
}
ch_reads_all
.filter { !it[2] }
.concat(ch_sra_fastq_ftp, ch_sra_fastq_dump)
.set { ch_reads_all }
}
ch_reads_all
.map { [ it[0].split('_')[0..-2].join('_'), it[1], it[4] ] }
.groupTuple(by: [0, 1])
.map { [ it[0], it[1], it[2].flatten() ] }
.set { ch_reads_all }
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/* -- -- */
/* -- MERGE RESEQUENCED FASTQ -- */
/* -- -- */
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/*
* STEP 2: Merge FastQ files with the same sample identifier
*/
process CAT_FASTQ {
tag "$sample"
input:
tuple val(sample), val(single_end), path(reads) from ch_reads_all
output:
tuple val(sample), val(single_end), path("*.merged.fastq.gz") into ch_cat_fastqc,
ch_cat_fastp
script:
readList = reads.collect{it.toString()}
if (!single_end) {
if (readList.size > 2) {
def read1 = []
def read2 = []
readList.eachWithIndex{ v, ix -> ( ix & 1 ? read2 : read1 ) << v }
"""
cat ${read1.sort().join(' ')} > ${sample}_1.merged.fastq.gz
cat ${read2.sort().join(' ')} > ${sample}_2.merged.fastq.gz
"""
} else {
"""
ln -s ${reads[0]} ${sample}_1.merged.fastq.gz
ln -s ${reads[1]} ${sample}_2.merged.fastq.gz
"""
}
} else {
if (readList.size > 1) {
"""
cat ${readList.sort().join(' ')} > ${sample}.merged.fastq.gz
"""
} else {
"""
ln -s $reads ${sample}.merged.fastq.gz
"""
}
}
}
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/* -- -- */
/* -- FASTQ QC -- */
/* -- -- */
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/*
* STEP 3: FastQC on input reads after merging libraries from the same sample
*/
process FASTQC {
tag "$sample"
label 'process_medium'
publishDir "${params.outdir}/preprocess/fastqc", mode: params.publish_dir_mode,
saveAs: { filename ->
filename.endsWith(".zip") ? "zips/$filename" : filename
}
when:
!params.skip_fastqc
input:
tuple val(sample), val(single_end), path(reads) from ch_cat_fastqc
output:
path "*.{zip,html}" into ch_fastqc_raw_reports_mqc
script:
"""
fastqc --quiet --threads $task.cpus *.fastq.gz
"""
}
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/* -- -- */
/* -- ADAPTER TRIMMING -- */
/* -- -- */
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/*
* STEP 4: Fastp adapter trimming and quality filtering
*/
if (!params.skip_adapter_trimming) {
process FASTP {
tag "$sample"
label 'process_medium'
publishDir "${params.outdir}/preprocess/fastp", mode: params.publish_dir_mode,
saveAs: { filename ->
if (filename.endsWith(".json")) filename
else if (filename.endsWith(".fastp.html")) filename
else if (filename.endsWith("_fastqc.html")) "fastqc/$filename"
else if (filename.endsWith(".zip")) "fastqc/zips/$filename"
else if (filename.endsWith(".log")) "log/$filename"
else params.save_trimmed ? filename : null
}
when:
!params.skip_variants || !params.skip_assembly
input:
tuple val(sample), val(single_end), path(reads) from ch_cat_fastp
output:
tuple val(sample), val(single_end), path("*.trim.fastq.gz") into ch_fastp_bowtie2,
ch_fastp_cutadapt,
ch_fastp_kraken2
path "*.json" into ch_fastp_mqc
path "*_fastqc.{zip,html}" into ch_fastp_fastqc_mqc
path "*.{log,fastp.html}"
path "*.fail.fastq.gz"
script:
// Added soft-links to original fastqs for consistent naming in MultiQC
autodetect = single_end ? "" : "--detect_adapter_for_pe"
"""
IN_READS='--in1 ${sample}.fastq.gz'
OUT_READS='--out1 ${sample}.trim.fastq.gz --failed_out ${sample}.fail.fastq.gz'
if $single_end; then
[ ! -f ${sample}.fastq.gz ] && ln -s $reads ${sample}.fastq.gz
else
[ ! -f ${sample}_1.fastq.gz ] && ln -s ${reads[0]} ${sample}_1.fastq.gz
[ ! -f ${sample}_2.fastq.gz ] && ln -s ${reads[1]} ${sample}_2.fastq.gz
IN_READS='--in1 ${sample}_1.fastq.gz --in2 ${sample}_2.fastq.gz'
OUT_READS='--out1 ${sample}_1.trim.fastq.gz --out2 ${sample}_2.trim.fastq.gz --unpaired1 ${sample}_1.fail.fastq.gz --unpaired2 ${sample}_2.fail.fastq.gz'
fi
fastp \\
\$IN_READS \\
\$OUT_READS \\
$autodetect \\
--cut_front \\
--cut_tail \\
--cut_mean_quality $params.cut_mean_quality \\
--qualified_quality_phred $params.qualified_quality_phred \\
--unqualified_percent_limit $params.unqualified_percent_limit \\
--length_required $params.min_trim_length \\
--trim_poly_x \\
--thread $task.cpus \\
--json ${sample}.fastp.json \\
--html ${sample}.fastp.html \\
2> ${sample}.fastp.log
fastqc --quiet --threads $task.cpus *.trim.fastq.gz
"""
}
} else {
ch_cat_fastp
.into { ch_fastp_bowtie2
ch_fastp_cutadapt
ch_fastp_kraken2 }
ch_fastp_mqc = Channel.empty()
ch_fastp_fastqc_mqc = Channel.empty()
}
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/* -- -- */
/* -- VARIANT CALLING PROCESSES -- */
/* -- -- */
///////////////////////////////////////////////////////////////////////////////
///////////////////////////////////////////////////////////////////////////////
/*
* PREPROCESSING: Build Bowtie2 index for viral genome
*/
process BOWTIE2_INDEX {
tag "$fasta"
label 'process_medium'
if (params.save_reference) {
publishDir "${params.outdir}/genome", mode: params.publish_dir_mode
}
when:
!params.skip_variants
input:
path fasta from ch_fasta
output:
path "Bowtie2Index" into ch_index
script:
"""
bowtie2-build \\
--seed 1 \\
--threads $task.cpus \\
$fasta \\
$index_base
mkdir Bowtie2Index && mv ${index_base}* Bowtie2Index
"""
}
/*
* PREPROCESSING: Build SnpEff database for viral genome
*/
process MAKE_SNPEFF_DB {
tag "${index_base}.fa"
label 'process_low'
if (params.save_reference) {
publishDir "${params.outdir}/genome", mode: params.publish_dir_mode
}
when:
(!params.skip_variants || !params.skip_assembly) && params.gff && !params.skip_snpeff
input:
path ("SnpEffDB/genomes/${index_base}.fa") from ch_fasta
path ("SnpEffDB/${index_base}/genes.gff") from ch_gff
output:
tuple path("SnpEffDB"), path("*.config") into ch_snpeff_db_varscan2,
ch_snpeff_db_ivar,
ch_snpeff_db_bcftools,
ch_snpeff_db_spades,
ch_snpeff_db_metaspades,
ch_snpeff_db_unicycler,
ch_snpeff_db_minia
script:
"""
echo "${index_base}.genome : ${index_base}" > snpeff.config
snpEff build -config snpeff.config -dataDir ./SnpEffDB -gff3 -v ${index_base}
"""
}
/*
* STEP 5.1: Map read(s) with Bowtie 2
*/
process BOWTIE2 {
tag "$sample"
label 'process_medium'
publishDir "${params.outdir}/variants/bam", mode: params.publish_dir_mode,
saveAs: { filename ->
if (filename.endsWith(".log")) "log/$filename"
else params.save_align_intermeds ? filename : null
}
when:
!params.skip_variants
input:
tuple val(sample), val(single_end), path(reads) from ch_fastp_bowtie2
path index from ch_index
output:
tuple val(sample), val(single_end), path("*.bam") into ch_bowtie2_bam
path "*.log" into ch_bowtie2_mqc
script:
input_reads = single_end ? "-U $reads" : "-1 ${reads[0]} -2 ${reads[1]}"
filter = params.filter_unmapped ? "-F4" : ""
"""
bowtie2 \\
--threads $task.cpus \\
--local \\
--very-sensitive-local \\
-x ${index}/${index_base} \\
$input_reads \\
2> ${sample}.bowtie2.log \\
| samtools view -@ $task.cpus -b -h -O BAM -o ${sample}.bam $filter -
"""
}
/*
* STEP 5.2: Convert BAM to coordinate sorted BAM
*/
process SORT_BAM {
tag "$sample"
label 'process_medium'
publishDir "${params.outdir}/variants/bam", mode: params.publish_dir_mode,
saveAs: { filename ->
if (filename.endsWith(".flagstat")) "samtools_stats/$filename"
else if (filename.endsWith(".idxstats")) "samtools_stats/$filename"
else if (filename.endsWith(".stats")) "samtools_stats/$filename"
else (params.protocol != 'amplicon' && params.skip_markduplicates) || params.save_align_intermeds ? filename : null
}
when:
!params.skip_variants
input:
tuple val(sample), val(single_end), path(bam) from ch_bowtie2_bam
output:
tuple val(sample), val(single_end), path("*.sorted.{bam,bam.bai}"), path("*.flagstat") into ch_sort_bam
path "*.{flagstat,idxstats,stats}" into ch_sort_bam_flagstat_mqc
script:
"""
samtools sort -@ $task.cpus -o ${sample}.sorted.bam -T $sample $bam
samtools index ${sample}.sorted.bam
samtools flagstat ${sample}.sorted.bam > ${sample}.sorted.bam.flagstat
samtools idxstats ${sample}.sorted.bam > ${sample}.sorted.bam.idxstats
samtools stats ${sample}.sorted.bam > ${sample}.sorted.bam.stats
"""
}
// Get total number of mapped reads from flagstat file
def get_mapped_from_flagstat(flagstat) {
def mapped = 0
flagstat.eachLine { line ->
if (line.contains(' mapped (')) {
mapped = line.tokenize().first().toInteger()
}
}
return mapped
}
// Function that checks the number of mapped reads from flagstat output
// and returns true if > params.min_mapped_reads and otherwise false
pass_mapped_reads = [:]
fail_mapped_reads = [:]
def check_mapped(sample,flagstat,min_mapped_reads=500) {
mapped = get_mapped_from_flagstat(flagstat)
c_reset = params.monochrome_logs ? '' : "\033[0m";
c_green = params.monochrome_logs ? '' : "\033[0;32m";
c_red = params.monochrome_logs ? '' : "\033[0;31m";
if (mapped < min_mapped_reads.toInteger()) {
log.info ">${c_red}>>>> $sample FAILED MAPPED READ THRESHOLD: ${mapped} < ${params.min_mapped_reads}. IGNORING FOR FURTHER DOWNSTREAM ANALYSIS! <<<<${c_reset}<"
fail_mapped_reads[sample] = mapped
return false
} else {
//log.info "-${c_green} Passed mapped read threshold > bowtie2 ($sample) >> ${mapped} <<${c_reset}"
pass_mapped_reads[sample] = mapped
return true
}
}
// Remove samples that failed mapped read threshold
ch_sort_bam
.filter { sample, single_end, bam, flagstat -> check_mapped(sample,flagstat,params.min_mapped_reads) }
.map { it[0..2] }
.set { ch_sort_bam }
/*
* STEP 5.3: Trim amplicon sequences with iVar
*/
if (params.protocol != 'amplicon') {
ch_sort_bam
.set { ch_ivar_trim_bam }
ch_ivar_trim_flagstat_mqc = Channel.empty()
ch_ivar_trim_log_mqc = Channel.empty()
} else {
process IVAR_TRIM {
tag "$sample"
label 'process_medium'
publishDir "${params.outdir}/variants/bam", mode: params.publish_dir_mode,
saveAs: { filename ->
if (filename.endsWith(".flagstat")) "samtools_stats/$filename"
else if (filename.endsWith(".idxstats")) "samtools_stats/$filename"
else if (filename.endsWith(".stats")) "samtools_stats/$filename"
else if (filename.endsWith(".log")) "log/$filename"
else params.skip_markduplicates || params.save_align_intermeds ? filename : null
}
when:
!params.skip_variants
input:
tuple val(sample), val(single_end), path(bam) from ch_sort_bam
path bed from ch_amplicon_bed