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pipeline.nf
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#!/usr/bin/env nextflow
def helpMessage() {
log.info"""
Usage:
The typical command for running the pipeline is as follows:
nextflow run federicacitarrella/pipelineGeneFusions --rnareads '*_{1,2}.fastq.gz' --dnareads_tumor '*_{3,4}.fastq.gz' --dnareads_normal '*_{5,6}.fastq.gz' -profile docker
Principal arguments:
--rnareads [file] Path to input RNA data (must be surrounded with quotes)
Format: fastq
--dnareads_tumor [file] Path to input tumor DNA data (must be surrounded with quotes)
Format: fastq, bam
--dnareads_normal [file] Path to input normal DNA data (must be surrounded with quotes)
Format: fastq, bam
-profile [str] Configuration profile to use.
Available: docker, local, test_docker, test_local
Tool flags:
--arriba [bool] Run Arriba (RNA)
--ericscript [bool] Run Ericscript (RNA)
--fusioncatcher [bool] Run FusionCatcher (RNA)
--integrate [bool] Run Integrate (RNA; RNA+DNA)
--genefuse [bool] Run GeneFuse (DNA)
References:
--ericscript_ref [file] Path to EricScript reference
--arriba_ref [dir] Path to Arriba reference
--fusioncatcher_ref [dir] Path to FusionCatcher reference
--integrate_ref [file] Path to Integrate reference
--genefuse_ref [file] Path to GeneFuse reference
Options:
--dnabam [bool] Specifies that the wgs input has bam format
--nthreads [int] Specifies the number of threads [8]
Other options:
--outdir [dir] The output directory where the results will be saved
""".stripIndent()
}
/*
================================================================================
SET UP CONFIGURATION VARIABLES
================================================================================
*/
// Show help message
if (params.help) {
helpMessage()
exit 0
}
// Files and folders set up from default directories or directories defined in command line
refDir_refgen = file(params.referenceGenome)
refDir_refgen_index = file(params.referenceGenome_index)
refFile_ericscript = file(params.ericscript_ref)
refDir_arriba = file(params.arriba_ref)
refDir_fusioncatcher = file(params.fusioncatcher_ref)
refDir_integrate = file(params.integrate_ref)
refDir_integrate_bwts = file(params.integrate_bwts)
refDir_genefuse = file(params.genefuse_ref)
// Skip variables set up to verify the existence of files and folders and eventually skip the download processes execution
params.skip_refgen = refDir_refgen.exists()
params.skip_refgen_index = refDir_refgen_index.exists()
params.skip_ericscript = refFile_ericscript.exists()
params.skip_arriba = refDir_arriba.exists()
params.skip_fusioncatcher= refDir_fusioncatcher.exists()
params.skip_integrate = refDir_integrate.exists()
params.skip_integrate_bulder = refDir_integrate_bwts.exists()
params.skip_genefuse = refDir_genefuse.exists()
// INTEGRATE variables set up (this variable could be modified, for this reason they cannot be defined in the configuration file)
integrateWGSt = false
integrateWGSn = false
command1 = ""
command2 = ""
// If the user inserts DNA data in the command line the INTEGRATE variable are modified
if (params.dnareads_tumor) {
integrateWGSt = true
command1 = "dna.tumor.bam"
}
if (params.dnareads_normal) {
integrateWGSn = true
command2 = "dna.normal.bam"
}
// RNA data channels creation
( rna_reads_ericscript , rna_reads_arriba , rna_reads_fusioncatcher , rna_reads_integrate , support1 , support2 ) = ( params.rnareads ? [
Channel.fromFilePairs(params.rnareads),
Channel.fromFilePairs(params.rnareads),
Channel.fromFilePairs(params.rnareads),
Channel.fromFilePairs(params.rnareads),
Channel.fromFilePairs(params.rnareads),
Channel.fromFilePairs(params.rnareads)
] : [
Channel.empty(),
Channel.empty(),
Channel.empty(),
Channel.empty(),
Channel.empty(),
Channel.empty(),
Channel.empty()
])
// DNA data channels creation
(dna_reads_tumor_integrate , dna_reads_tumor_genefuse ) = ( params.dnareads_tumor ? [Channel.fromFilePairs(params.dnareads_tumor, size: -1 ), Channel.fromFilePairs(params.dnareads_tumor, size: -1 )] : [support1.map{id,reads -> tuple(id,"1")}, Channel.empty()] ) // support1 channel is used to keep the integrate_concerter process running even if DNA data are not provided
(dna_reads_normal_integrate) = ( params.dnareads_normal ? [Channel.fromFilePairs(params.dnareads_normal, size: -1 )] : [support2.map{id,reads -> tuple(id,"1")}] )
Channel.fromPath(params.referenceGenome).into{ input_ch1_refgen ; input_ch2_refgen ; input_ch3_refgen ; input_ch4_refgen ; input_ch5_refgen}
Channel.fromPath(params.referenceGenome_index).set{ input_ch1_refgen_index }
Channel.fromPath(params.ericscript_ref).set{ input_ch_ericscript }
Channel.fromPath(params.arriba_ref).set{ input_ch_arriba }
Channel.fromPath(params.fusioncatcher_ref).set{ input_ch_fusioncatcher }
Channel.fromPath(params.integrate_ref).into{ input_ch1_integrate;input_ch2_integrate;input_ch3_integrate }
Channel.fromPath(params.integrate_bwts).set{ input_ch1_bwts }
Channel.fromPath(params.genefuse_ref).set{ input_ch1_genefuse }
(refgen_downloader , refgen_integrate , refgen_integrate_builder, refgen_integrate_converter, refgen_genefuse, refgen_referenceGenome_index) = ( params.skip_refgen ? [Channel.empty(), input_ch1_refgen, input_ch2_refgen, input_ch3_refgen, input_ch4_refgen, input_ch5_refgen] : [input_ch1_refgen, Channel.empty(), Channel.empty(), Channel.empty(), Channel.empty(), Channel.empty()] )
(refgen_index_trigger , refgen_index) = ( (params.skip_refgen_index || params.dnabam) ? [Channel.empty(), input_ch1_refgen_index] : [input_ch1_refgen_index, Channel.empty()] )
(ch1_ericscript, ch2_ericscript) = ( params.skip_ericscript ? [Channel.empty(), input_ch_ericscript] : [input_ch_ericscript, Channel.empty()] )
(ch1_arriba, ch2_arriba) = ( params.skip_arriba ? [Channel.empty(), input_ch_arriba] : [input_ch_arriba, Channel.empty()] )
(ch1_fusioncatcher , ch2_fusioncatcher) = ( params.skip_fusioncatcher ? [Channel.empty(), input_ch_fusioncatcher] : [input_ch_fusioncatcher, Channel.empty()] )
(ch1_integrate , ch2_integrate , ch3_integrate, ch4_integrate) = ( params.skip_integrate ? [Channel.empty(), input_ch1_integrate, input_ch2_integrate, input_ch3_integrate] : [input_ch1_integrate, Channel.empty(), Channel.empty(), Channel.empty()] )
(ch1_integrate_bwts , ch2_integrate_bwts) = ( params.skip_integrate_bulder ? [Channel.empty(), input_ch1_bwts] : [input_ch1_bwts, Channel.empty()] )
(ch1_genefuse , ch2_genefuse ) = ( params.skip_genefuse ? [Channel.empty(), input_ch1_genefuse] : [input_ch1_genefuse, Channel.empty()] )
/*
* Reference Genome
*/
process referenceGenome_downloader{
// Tag shown on the terminal while the process is running
tag "Downloading"
// publishDir publishes the output in a specific folder with copy mode
publishDir "${params.outdir}/reference_genome", mode: 'copy'
input:
val trigger from refgen_downloader
output:
file "hg38.fa" into refgen_integrate_builder_down, refgen_integrate_converter_down, refgen_referenceGenome_index_down, refgen_integrate_down, refgen_genefuse_down
// Conditions for the process execution
when: params.integrate || params.genefuse || !(params.arriba || params.ericscript || params.fusioncatcher || params.genefuse || params.integrate)
script:
"""
#!/bin/bash
export PATH="${params.envPath_integrate}:$PATH"
curl -O -J -L https://osf.io/yevub/download
"""
}
//gdown "https://drive.google.com/uc?export=download&confirm=qgOc&id=1AfNX3UvUOn4kSsu-ECrYChq8F8yJbFCI"
process referenceGenome_index{
tag "Downloading"
publishDir "${params.outdir}/reference_genome", mode: 'copy'
input:
val x from refgen_index_trigger
file refgen from refgen_referenceGenome_index.mix(refgen_referenceGenome_index_down)
output:
file "index" into refgen_index_down
when: params.integrate || !(params.arriba || params.ericscript || params.fusioncatcher || params.genefuse || params.integrate)
shell:
'''
#!/bin/bash
export PATH="!{params.envPath_integrate}:$PATH"
mkdir index && cd "$_"
cp ../!{refgen} .
bwa index hg38.fa
'''
}
/*
================================================================================
FUSION PIPELINE
================================================================================
*/
/*
* EricScript
*/
process ericsctipt_downloader{
tag "Downloading"
publishDir "${params.outdir}/ericscript/files", mode: 'copy'
input:
val x from ch1_ericscript
output:
file "ericscript_db_homosapiens_ensembl84" into ch3_ericscript
when: params.ericscript || !(params.arriba || params.ericscript || params.fusioncatcher || params.genefuse || params.integrate)
script:
"""
#!/bin/bash
export PATH="${params.envPath_ericscript}:$PATH"
curl -O -J -L https://osf.io/54s6h/download
tar -xf ericscript_db_homosapiens_ensembl84.tar.bz2
rm ericscript_db_homosapiens_ensembl84.tar.bz2
"""
}
//gdown "https://drive.google.com/uc?export=download&confirm=qgOc&id=1VENACpUv_81HbIB8xZN0frasrAS7M4SP"
process ericscript{
tag "${pair_id}"
publishDir "${params.outdir}/ericscript", mode: 'copy'
input:
tuple pair_id, file(rna_reads), file(ericscript_db) from rna_reads_ericscript.combine(ch2_ericscript.mix(ch3_ericscript))
output:
file "output/${pair_id}" optional true into ericscript_fusions
when: params.ericscript || !(params.arriba || params.ericscript || params.fusioncatcher || params.genefuse || params.integrate)
script:
reads = "../${rna_reads[0]} ../${rna_reads[1]}" //reads = params.single_end ? rna_reads[0] : "../${rna_reads[0]} ../${rna_reads[1]}"
"""
#!/bin/bash
export PATH="${params.envPath_ericscript}:$PATH"
mkdir output && cd output
ericscript.pl -o ./${pair_id} -db ../${ericscript_db} ${reads}
"""
}
/*
* Arriba
*/
process arriba_downloader{
tag "Downloading"
publishDir "${params.outdir}/arriba", mode: 'copy'
input:
val x from ch1_arriba
output:
file "files" into ch3_arriba
when: params.arriba || !(params.arriba || params.ericscript || params.fusioncatcher || params.genefuse || params.integrate)
shell:
'''
#!/bin/bash
export PATH="!{params.envPath_arriba}bin:$PATH"
mkdir files && cd "$_"
!{params.envPath_arriba}var/lib/arriba/download_references.sh GRCh38+ENSEMBL93
'''
}
process arriba{
tag "${pair_id}"
publishDir "${params.outdir}/arriba", mode: 'copy'
input:
tuple pair_id, file(rna_reads), file(arriba_ref) from rna_reads_arriba.combine(ch2_arriba.mix(ch3_arriba))
output:
file "output/${pair_id}" optional true into arriba_fusions
when: params.arriba || !(params.arriba || params.ericscript || params.fusioncatcher || params.genefuse || params.integrate)
script:
"""
#!/bin/bash
export PATH="${params.envPath_arriba}bin:$PATH"
run_arriba.sh ${arriba_ref}/STAR_index_GRCh38_ENSEMBL93/ ${arriba_ref}/ENSEMBL93.gtf ${arriba_ref}/GRCh38.fa ${params.envPath_arriba}var/lib/arriba/blacklist_hg19_hs37d5_GRCh37_v2.1.0.tsv.gz ${params.envPath_arriba}var/lib/arriba/known_fusions_hg19_hs37d5_GRCh37_v2.1.0.tsv.gz ${params.envPath_arriba}var/lib/arriba/protein_domains_hg19_hs37d5_GRCh37_v2.1.0.gff3 ${params.nthreads} ${rna_reads}
mkdir output && mkdir output/${pair_id}
mv *.out output/${pair_id}
mv *.tsv output/${pair_id}
mv *.out output/${pair_id}
mv *bam* output/${pair_id}
"""
}
/*
* FusionCatcher
*/
process fusioncatcher_downloader{
tag "Downloading"
publishDir "${params.outdir}/fusioncatcher", mode: 'copy'
input:
val x from ch1_fusioncatcher
output:
file "files" into ch3_fusioncatcher
when: params.fusioncatcher || !(params.arriba || params.ericscript || params.fusioncatcher || params.genefuse || params.integrate)
shell:
'''
#!/bin/bash
mkdir -p files && cd "$_"
wget http://sourceforge.net/projects/fusioncatcher/files/data/human_v102.tar.gz.aa
wget http://sourceforge.net/projects/fusioncatcher/files/data/human_v102.tar.gz.ab
wget http://sourceforge.net/projects/fusioncatcher/files/data/human_v102.tar.gz.ac
wget http://sourceforge.net/projects/fusioncatcher/files/data/human_v102.tar.gz.ad
cat human_v102.tar.gz.* | tar xz
ln -s human_v102 current
'''
}
process fusioncatcher{
tag "${pair_id}"
publishDir "${params.outdir}/fusioncatcher", mode: 'copy'
input:
tuple pair_id, file(rna_reads), file(fusioncatcher_db) from rna_reads_fusioncatcher.combine(ch2_fusioncatcher.mix(ch3_fusioncatcher))
output:
file "output/${pair_id}" optional true into fusioncatcher_fusions
when: params.fusioncatcher || !(params.arriba || params.ericscript || params.fusioncatcher || params.genefuse || params.integrate)
script:
reads = "${rna_reads[0]},${rna_reads[1]}" //reads = params.single_end ? rna_reads[0] : "${rna_reads[0]},${rna_reads[1]}"
"""
#!/bin/bash
export PATH="${params.envPath_fusioncatcher}:$PATH"
fusioncatcher -d ${fusioncatcher_db}/human_v102 -i ${reads} -o output/${pair_id}
"""
}
/*
* INTEGRATE
*/
process integrate_downloader{
tag "Downloading"
publishDir "${params.outdir}/integrate", mode: 'copy'
input:
val x from ch1_integrate
output:
file "files" into ch5_integrate, ch6_integrate, ch7_integrate
when: params.integrate || !(params.arriba || params.ericscript || params.fusioncatcher || params.genefuse || params.integrate)
shell:
'''
#!/bin/bash
export PATH="!{params.envPath_integrate}:$PATH"
mkdir files && cd "$_"
wget https://genome-idx.s3.amazonaws.com/bt/GRCh38_noalt_as.zip
unzip GRCh38_noalt_as.zip
rm GRCh38_noalt_as.zip
curl -O -J -L https://osf.io/dgvcx/download
curl -O -J -L https://osf.io/gv7sq/download
tar -xvf INTEGRATE.0.2.6.tar.gz
rm INTEGRATE.0.2.6.tar.gz
cd INTEGRATE_0_2_6 && mkdir INTEGRATE-build && cd "$_"
cmake ../Integrate/ -DCMAKE_BUILD_TYPE=release
make
'''
}
//gdown "https://drive.google.com/uc?export=download&confirm=qgOc&id=18SUV1abrk_MhYGOG6kzJPIeIJ5Zs5Yvb"
//gdown "https://drive.google.com/uc?export=download&confirm=qgOc&id=14VCiEYWCl5m9bo_tsvNGQDUNUgtLje9Y"
process integrate_builder{
tag "Building"
publishDir "${params.outdir}/integrate/files", mode: 'copy'
input:
val x from ch1_integrate_bwts
file refgen from refgen_integrate_builder.mix(refgen_integrate_builder_down)
file integrate_db from ch4_integrate.mix(ch7_integrate)
output:
file "bwts" into ch8_integrate
when: params.integrate || !(params.arriba || params.ericscript || params.fusioncatcher || params.genefuse || params.integrate)
shell:
'''
#!/bin/bash
export PATH="!{params.envPath_integrate}:$PATH"
LD_LIBRARY_PATH=/usr/local/lib
LD_LIBRARY_PATH=$LD_LIBRARY_PATH:!{integrate_db}/INTEGRATE_0_2_6/INTEGRATE-build/vendor/src/libdivsufsort-2.0.1-build/lib/
export LD_LIBRARY_PATH
mkdir ./bwts
!{integrate_db}/INTEGRATE_0_2_6/INTEGRATE-build/bin/Integrate mkbwt !{refgen}
'''
}
process integrate_converter{
tag "${pair_id}"
publishDir "${params.outdir}/integrate", mode: 'copy'
input:
tuple pair_id, file(rna_reads), file(integrate_db), file(refgen), file(index), file(wgstinput), file(wgsninput) from rna_reads_integrate.combine(ch2_integrate.mix(ch5_integrate)).combine(refgen_integrate_converter.mix(refgen_integrate_converter_down)).combine(refgen_index.mix(refgen_index_down)).join(dna_reads_tumor_integrate).join(dna_reads_normal_integrate)
output:
tuple pair_id, file("input/${pair_id}") into integrate_input
when: params.integrate || !(params.arriba || params.ericscript || params.fusioncatcher || params.genefuse || params.integrate)
script:
"""
#!/bin/bash
export PATH="${params.envPath_integrate}:$PATH"
tophat --no-coverage-search ${integrate_db}/GRCh38_noalt_as/GRCh38_noalt_as ${rna_reads}
mkdir input && mkdir input/${pair_id}
cp tophat_out/accepted_hits.bam input/${pair_id}
cp tophat_out/unmapped.bam input/${pair_id}
if ${params.dnabam}; then
if ${integrateWGSt}; then
cp ${wgstinput} input/${pair_id}/dna.tumor.bam
fi
if ${integrateWGSn}; then
cp ${wgsninput} input/${pair_id}/dna.normal.bam
fi
elif ${integrateWGSt} || ${integrateWGSn}; then
mkdir index_dir
cp ${index}/* index_dir
if ${integrateWGSt}; then
bwa mem index_dir/hg38.fa ${wgstinput} | samtools sort -o input/${pair_id}/dna.tumor.bam
fi
if ${integrateWGSn}; then
bwa mem index_dir/hg38.fa ${wgsninput} | samtools sort -o input/${pair_id}/dna.normal.bam
fi
fi
"""
}
process integrate{
tag "${pair_id}"
publishDir "${params.outdir}/integrate", mode: 'copy'
input:
tuple pair_id, file(input), file(integrate_db), file(refgen), file(bwts) from integrate_input.combine(ch3_integrate.mix(ch6_integrate)).combine(refgen_integrate.mix(refgen_integrate_down)).combine(ch2_integrate_bwts.mix(ch8_integrate))
output:
file "output/${pair_id}" optional true into integrate_fusions
when: params.integrate || !(params.arriba || params.ericscript || params.fusioncatcher || params.genefuse || params.integrate)
shell:
'''
#!/bin/bash
export PATH="!{params.envPath_integrate}:$PATH"
cp !{input}/* .
parallel samtools index ::: *.bam
LD_LIBRARY_PATH=/usr/local/lib
LD_LIBRARY_PATH=$LD_LIBRARY_PATH:!{integrate_db}/INTEGRATE_0_2_6/INTEGRATE-build/vendor/src/libdivsufsort-2.0.1-build/lib/
export LD_LIBRARY_PATH
!{integrate_db}/INTEGRATE_0_2_6/INTEGRATE-build/bin/Integrate fusion !{refgen} !{integrate_db}/annot.refseq.txt !{bwts} accepted_hits.bam unmapped.bam !{command1} !{command2}
mkdir output && mkdir output/!{pair_id}
cp *.tsv output/!{pair_id}
cp *.txt output/!{pair_id}
'''
}
/*
* GeneFuse
*/
process genefuse_downloader{
tag "Downloading"
publishDir "${params.outdir}/genefuse", mode: 'copy'
input:
val x from ch1_genefuse
output:
file "files" into ch3_genefuse
when: params.genefuse || !(params.arriba || params.ericscript || params.fusioncatcher || params.genefuse || params.integrate)
shell:
'''
#!/bin/bash
export PATH="!{params.envPath_integrate}:$PATH"
mkdir files && cd "$_"
curl -O -J -L https://osf.io/8r9fh/download
chmod a+x ./genefuse
curl -O -J -L https://osf.io/jqywz/download
'''
}
//gdown "https://drive.google.com/uc?export=download&confirm=qgOc&id=1OBLTo-yGZ88UGcF0F3v_7n8mLTQblWg8"
//gdown "https://drive.google.com/uc?export=download&confirm=qgOc&id=1eRI5lAw0qntj0EbEpaNpvRA7saw_iyY3"
process genefuse_converter{
tag "${pair_id}"
publishDir "${params.outdir}/genefuse", mode: 'copy'
input:
tuple pair_id, file(wgstinput) from dna_reads_tumor_genefuse
output:
tuple pair_id, file("input/${pair_id}") into genefuse_input
when: params.genefuse || !(params.arriba || params.ericscript || params.fusioncatcher || params.genefuse || params.integrate)
script:
"""
#!/bin/bash
export PATH="${params.envPath_genefuse}:$PATH"
mkdir input && mkdir input/${pair_id}
if ${params.dnabam} && ${integrateWGSt}; then
samtools sort -n -o ${wgstinput}
samtools fastq -@ ${params.nthreads} ${wgstinput} -1 ${pair_id}_3.fq.gz -2 ${pair_id}_4.fq.gz -0 /dev/null -s /dev/null -n
cp *.fq.gz input/${pair_id}
elif ${integrateWGSt}; then
cp ${wgstinput} input/${pair_id}
fi
"""
}
process genefuse{
tag "${pair_id}"
publishDir "${params.outdir}/genefuse", mode: 'copy'
input:
tuple pair_id, file(input), file(refgen), file(genefuse_db) from genefuse_input.combine(refgen_genefuse.mix(refgen_genefuse_down)).combine(ch2_genefuse.mix(ch3_genefuse))
output:
file "output/${pair_id}" optional true into genefuse_fusions
when: params.genefuse || !(params.arriba || params.ericscript || params.fusioncatcher || params.genefuse || params.integrate)
script:
"""
#!/bin/bash
export PATH="${params.envPath_genefuse}:$PATH"
cp ${input}/* .
${genefuse_db}/genefuse -r ${refgen} -f ${genefuse_db}/druggable.hg38.csv -1 ${pair_id}_3.* -2 ${pair_id}_4.* -h report.html > result
mkdir output && mkdir output/${pair_id}
cp report.html output/${pair_id}
cp result output/${pair_id}
"""
}