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main.nf
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#!/usr/bin/env nextflow
/*
*
======================================================================
ViPR: Viral amplicon/enrichment analysis and intrahost variant calling
======================================================================
# Homepage / Documentation
https://github.com/nf-core/vipr
# Authors
Andreas Wilm <wilma@gis.a-star.edu.sg>
-------------------------------------------------------------------------------
*/
def helpMessage() {
log.info"""
===================================
nfcore/vipr ~ version ${params.pipelineVersion}
===================================
Usage:
The typical command for running the pipeline is as follows:
nextflow run nf-core/vipr --reads '*_R{1,2}.fastq.gz' -profile standard,docker
Mandatory arguments:
--reads Path to input data (must be surrounded with quotes)
-profile Configuration profile to use. uppmax / uppmax_modules / hebbe / docker / aws
Other options:
--outdir The output directory where the results will be saved
-w/--work-dir The temporary directory where intermediate data will be saved
--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
--maxMultiqcEmailFileSize 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)
AWSBatch options:
--awsqueue The AWSBatch JobQueue that needs to be set when running on AWSBatch
--awsregion The AWS Region for your AWS Batch job to run on
""".stripIndent()
}
/*
* SET UP CONFIGURATION VARIABLES
*/
// Show help emssage
params.help = false
if (params.help){
helpMessage()
exit 0
}
//AWSBatch sanity checking
if(workflow.profile == 'awsbatch'){
if (!params.awsqueue || !params.awsregion) exit 1, "Specify correct --awsqueue and --awsregion parameters on AWSBatch!"
if (!workflow.workDir.startsWith('s3') || !params.outdir.startsWith('s3')) exit 1, "Specify S3 URLs for workDir and outdir parameters on AWSBatch!"
}
// Check that Nextflow version is up to date enough
try {
if( ! nextflow.version.matches(">= $params.nf_required_version") ){
throw GroovyException("Nextflow version too old: ${workflow.nextflow.version} < $params.nf_required_version")
}
} catch (all) {
log.error "====================================================\n" +
" Nextflow version $params.nf_required_version required! You are running v$workflow.nextflow.version.\n" +
" Pipeline execution will continue, but things may break.\n" +
" Please run `nextflow self-update` to update Nextflow.\n" +
"============================================================"
}
/* Input validation
*/
input_ref_fasta = file(params.ref_fasta)
if (!input_ref_fasta.exists())
exit 1, "Missing input reference fasta file: ${input_ref_fasta}"
cont_fasta = file(params.cont_fasta)
if (!cont_fasta.exists())
exit 1, "Missing contamination fasta file: ${cont_fasta}"
if(!params.skip_kraken) {
kraken_db = file(params.kraken_db)
if (!kraken_db.exists())
exit 1, "Missing contamination fasta file: ${kraken_db}"
}
log.info "=================================================="
log.info " nf-core/vipr : Viral amplicon/enrichment analysis and intrahost variant calling: v${params.pipelineVersion}"
log.info "=================================================="
def GetReadPair = { sk, rk ->
// FIXME if files don't exist, their path might be relative to the input yaml
// see https://gist.github.com/ysb33r/5804364
tuple(file(params.samples[sk].readunits[rk]['fq1']),
file(params.samples[sk].readunits[rk]['fq2']))
}
def GetReadUnitKeys = { sk ->
params.samples[sk].readunits.keySet()
}
/* input channel for references used in decontamination step
*/
cont_fasta_ch = Channel.from(
file(cont_fasta), file(cont_fasta + ".amb"), file(cont_fasta + ".ann"),
file(cont_fasta + ".bwt"), file(cont_fasta + ".pac"),
file(cont_fasta + ".sa")).toList()
//cont_fasta_ch.subscribe { println "cont_fasta_ch $it" }
/* FIXME allow other means of defining input, e.g. CSV.
* input for fastq files. channel has sample name as key and all read pairs following
* see https://groups.google.com/forum/#!topic/nextflow/CF7Joh5xrkU
*/
sample_keys = params.samples.keySet()
println "List of samples: " + sample_keys.join(", ")
Channel
.from(sample_keys)
.map { sk -> tuple(sk, GetReadUnitKeys(sk).collect{GetReadPair(sk, it)}.flatten()) }
.set { fastq_ch }
//fastq_ch.subscribe { println "$it" }
/* Trim and combine read-pairs per sample
*/
process trim_and_combine {
tag { "Preprocessing of " + reads.size()/2 + " read pairs for " + sample_id }
//publishDir "${params.outdir}/${sample_id}/reads/", mode: 'copy'
input:
set sample_id, file(reads) from fastq_ch
output:
set sample_id, file("${sample_id}_R1-trimmed.fastq.gz"), file("${sample_id}_R2-trimmed.fastq.gz") into trim_and_combine_ch
script:
"""
# loop over readunits in pairs per sample
pairno=0
echo ${reads.join(" ")} | xargs -n2 | while read fq1 fq2; do
let pairno=pairno+1
# note: don't make reads smaller than assembler kmer length
skewer --quiet -t ${task.cpus} -m pe -q 3 -n -l 31 -z -o pair\${pairno}-skewer-out \$fq1 \$fq2;
cat *-trimmed-pair1.fastq.gz >> ${sample_id}_R1-trimmed.fastq.gz;
cat *-trimmed-pair2.fastq.gz >> ${sample_id}_R2-trimmed.fastq.gz;
rm *-trimmed-pair[12].fastq.gz;
done
fastqc -t {task.cpus} ${sample_id}_R1-trimmed.fastq.gz ${sample_id}_R2-trimmed.fastq.gz;
"""
}
/* Decontaminate reads against host reference
*/
process decont {
tag { "Decontaminating " + sample_id }
publishDir "${params.outdir}/${sample_id}/reads/", mode: 'copy'
input:
set sample_id, file(fq1), file(fq2) from trim_and_combine_ch
set file(cont_fasta), file(cont_amb), file(cont_ann), file(cont_bwt), \
file(cont_pac), file(cont_sa) from cont_fasta_ch
output:
set sample_id, file("${sample_id}_trimmed_decont_1.fastq.gz"), file("${sample_id}_trimmed_decont_2.fastq.gz") into \
fastq_for_tadpole, fastq_for_polish_assembly_ch, fastq_for_mapping_ch, fastq_for_kraken_ch
set file("${sample_id}_trimmed_decont_1_fastqc.zip"), file("${sample_id}_trimmed_decont_2_fastqc.zip"), \
file("${sample_id}_trimmed_decont_1_fastqc.html"), file("${sample_id}_trimmed_decont_2_fastqc.html") into fastqc_ch
script:
// bbduk should be faster but uses plenty of memory (>32GB for human)
"""
decont.py -i ${fq1} ${fq2} -t ${task.cpus} -c 0.5 -r ${cont_fasta} -o ${sample_id}_trimmed_decont;
# since this is the last fastqc processing step, let's run fastqc here
fastqc -t {task.cpus} ${sample_id}_trimmed_decont_1.fastq.gz ${sample_id}_trimmed_decont_2.fastq.gz;
"""
}
/* Metagenomics classification: QC for sample purity
*/
if(!params.skip_kraken) {
process kraken {
tag { "Running Kraken on " + sample_id }
publishDir "${params.outdir}/${sample_id}/", mode: 'copy'
input:
set sample_id, file(fq1), file(fq2) from fastq_for_kraken_ch
output:
file("${sample_id}_kraken.report")
script:
"""
kraken --threads ${task.cpus} --preload --db ${kraken_db} \
-paired ${fq1} ${fq2} > kraken.out;
# do not gzip! otherwise kraken-report happily runs (with some warnings) and produces rubbish results
kraken-report --db ${kraken_db} kraken.out > ${sample_id}_kraken.report
"""
}
}
/* Assembly of reads. only few programs can assemble at the depth
* possible for such samples (>100k). tadpole gave consistently better
* results (fewer errors/variants) than spades (with diginorm)
*/
process tadpole {
tag { "Tadpole assembly of " + sample_id }
publishDir "${params.outdir}/${sample_id}/", mode: 'copy'
input:
set sample_id, file(fq1), file(fq2) from fastq_for_tadpole
output:
set sample_id, file("${sample_id}_contigs.fa") into contigs_ch
script:
"""
tadpole.sh -Xmx10g threads=${task.cpus} in=${fq1} in2=${fq2} out=${sample_id}_contigs.fa,
"""
}
/* Orient contigs according to reference and fill gaps with reference
*/
process gap_fill_assembly {
tag { "Orienting and gap filling contigs for " + sample_id }
publishDir "${params.outdir}/${sample_id}/", mode: 'copy'
// capture special error code, telling us that we cannot proceed for valid reasons.
// meaning of consequently missing downstream files needs to be reflected in docs
errorStrategy = { task.exitStatus == 3 ? 'ignore' : 'terminate' }
input:
set sample_id, file(contigs_fa) from contigs_ch
file(input_ref_fasta)
output:
set sample_id, file("${sample_id}-gap-filled-assembly.fa"), file("${sample_id}-gap-filled-assembly.gaps.bed") \
into gap_filled_assembly_ch
script:
"""
set +e;
log=${sample_id}-gap-filled-assembly.log;
simple_contig_joiner.py -c ${contigs_fa} -r ${input_ref_fasta} \
-s "${sample_id}-gap-filled-assembly" -o ${sample_id}-gap-filled-assembly.fa \
-b "${sample_id}-gap-filled-assembly.gaps.bed" >& \$log;
rc=\$?
if [ \$rc -ne 0 ]; then
# nothing to join, means we cannot continue. so stop here.
grep 'Nothing to join' \$log && exit 3;
exit \$rc;
fi
"""
}
/* Polish assembly by repeated mapping, variant calling and variant incorporation
*/
process polish_assembly {
tag { "Polishing assembly for " + sample_id }
publishDir "${params.outdir}/${sample_id}/", mode: 'copy'
input:
set sample_id, file(assembly_fa), file(assembly_gaps_bed), file(fq1), file(fq2) \
from gap_filled_assembly_ch.join(fastq_for_polish_assembly_ch)
output:
set sample_id, file("${sample_id}_polished_assembly.fa") into polished_assembly_ch
script:
"""
# downsample to 1M reads to increase runtime
seqtk sample -s 666 ${fq1} 1000000 | gzip > R1_ds.R1.fastq.gz;
seqtk sample -s 666 ${fq2} 1000000 | gzip > R2_ds.R2.fastq.gz;
polish_viral_ref.sh -t ${task.cpus} -1 R1_ds.R1.fastq.gz -2 R2_ds.R2.fastq.gz \
-r ${assembly_fa} -o ${sample_id}_polished_assembly.fa
"""
}
/* Mapping against polished assembly
*/
process final_mapping {
tag { "Mapping to polished assembly for " + sample_id }
publishDir "${params.outdir}/${sample_id}/", mode: 'copy'
input:
set sample_id, file(ref_fa), file(fq1), file(fq2) \
from polished_assembly_ch.join(fastq_for_mapping_ch)
output:
set sample_id, file(ref_fa), file("${sample_id}.bam"), file("${sample_id}.bam.bai") into \
final_mapping_for_vcf_ch, final_mapping_for_cov_ch
set sample_id, "${sample_id}.bam.stats" into final_mapping_bamstats_ch
script:
"""
bwa index ${ref_fa};
samtools faidx ${ref_fa};
bwa mem -t ${task.cpus} ${ref_fa} ${fq1} ${fq2} | \
lofreq viterbi -f ${ref_fa} - | \
lofreq alnqual -u - ${ref_fa} | \
lofreq indelqual --dindel -f ${ref_fa} - | \
samtools sort -o ${sample_id}.bam -T ${sample_id}.final.tmp -;
samtools index ${sample_id}.bam;
samtools stats ${sample_id}.bam > ${sample_id}.bam.stats
"""
}
/* Low frequency variant calling
*/
process var_calling {
tag { "Final variant calling for " + sample_id }
publishDir "${params.outdir}/${sample_id}/", mode: 'copy'
input:
set sample_id, file(ref_fa), file(bam), file(bai) from final_mapping_for_vcf_ch
output:
set sample_id, file(ref_fa), file("${sample_id}.vcf.gz") into vcf_ch
script:
"""
samtools faidx ${ref_fa};
lofreq call-parallel --pp-threads ${task.cpus} -f ${ref_fa} \
-d 1000000 --call-indels -o ${sample_id}.vcf.gz ${bam}
"""
}
/* Compute coverage
*
* FIXME: fast process, best joined with another process
*/
process genomecov {
tag { "Genome coverage for " + sample_id }
publishDir "${params.outdir}/${sample_id}/", mode: 'copy'
input:
set sample_id, file(ref_fa), file(bam), file(bai) from final_mapping_for_cov_ch
output:
set sample_id, file("${sample_id}.cov.gz") into cov_ch
script:
"""
# note: -d is one-based. -dz is zero-based but only non-zero values, so less explicit.
bedtools genomecov -d -ibam ${bam} | gzip > ${sample_id}.cov.gz;
"""
}
/* Plot coverage and variant AF. Also polish reference for upload
*/
process vipr_tools {
tag { "Plotting AF vs. coverage and readying fasta for " + sample_id }
publishDir "${params.outdir}/${sample_id}/", mode: 'copy'
input:
set sample_id, file(cov), file(ref_fa), file(vcf) from cov_ch.join(vcf_ch)
output:
set sample_id, file("${sample_id}_af-vs-cov.html"), file("${sample_id}_0cov2N.fa")
script:
"""
vipr_af_vs_cov_html.py --vcf ${vcf} --cov ${cov} --plot ${sample_id}_af-vs-cov.html;
vipr_gaps_to_n.py -i ${ref_fa} -c ${cov} > ${sample_id}_0cov2N.fa;
"""
}
/* Introspection
*
* https://www.nextflow.io/docs/latest/metadata.html
*/
workflow.onComplete {
println """Pipeline execution summary
---------------------------
Completed at : ${workflow.complete}
Duration : ${workflow.duration}
Success : ${workflow.success}
Work Dir : ${workflow.workDir}
Exit status : ${workflow.exitStatus}
Error report : ${workflow.errorReport ?: '-'}
"""
}
workflow.onError {
println "Oops... Pipeline execution stopped with the following message: ${workflow.errorMessage}"
}