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main.nf
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#!/usr/bin/env nextflow
/*
========================================================================================
MeRIPseqPipe
========================================================================================
MeRIPseqPipe：An integrated analysis pipeline for MeRIPseq data based on Nextflow.
#### Homepage / Documentation
https://github.com/canceromics/MeRIPseqPipe
----------------------------------------------------------------------------------------
*/
/*
* Authors:
* Bao Xiaoqiong <baoxq@sysucc.org.cn>
* Zhu Kaiyu <zhuky5@mail2.sysu.edu.cn>:
* Qi Zhao <zhaoqi@sysucc.org.cn>;
*/
/*
requirement:
- FastQC/fastp
- STAR/HISAT2/TopHat2/BWA
- SAMtools/RseQC
- MeTPeak/MACS2/MATK/Meyer
- RobustRankAggreg/MSPC/BEDTools
- featureCounts/DESeq2/EdgeR
- QNB
- HMOER
- igvtools
*/
def helpMessage() {
log.info"""
Usage:
The typical command for running the pipeline is as follows:
nextflow run path/to/MeRIPseqPipe --designfile 'path/to/designfile.tsv' --comparefile 'path/to/compare.txt' -profile <docker/test/conda> -resume
OR,
nextflow path/to/MeRIPseqPipe/main.nf -c path/to/MeRIPseqPipe/nextflow.config --designfile 'path/to/designfile/designfile.tsv' --comparefile 'path/to/comparefile/compare.txt' -profile <docker/test/conda> -resume
Mandatory arguments:
--designfile .tsv format(table splited by tabs): Sample_ID, input_R1, input_R2, ip_R1, ip_R2, Group_ID
--comparefile .txt format: control_vs_treated;
"false" for projects without differential analysis and "two_groups" for only two groups in the designfile
-profile Configuration files can contain the definition of one or more profiles.
Available: conda, docker, test
References If not specified in the configuration file or you wish to overwrite any of the references.
--fasta Path to Fasta reference
--gtf Path to GTF reference
Index:
--rRNA_fasta Path to HISAT2 rRNA fasta reference
--tophat2_index Path to TopHat2 index, eg. 'path/to/Tophat2Index/*'
--hisat2_index Path to HISAT2 index, eg. 'path/to/HISAT2Index/*'
--bwa_index Path to BWA index, eg. 'path/to/BWAIndex/*'
--star_index Path to STAR index, eg. 'path/to/STARIndex/*'
Main parameters of analysis mode:
--stranded "yes" OR "no" OR "reverse"
--mapq_cutoff [0-255] for filtering reads of different mapping quality
--featurecount_minMQS Integer giving the minimum mapping quality score a read must satisfy in order to be counted.
--motiflength length for HOMER motif searching
--delfc the threshold values of differential gene filtering
--dmlfc the threshold values of differential peak filtering
--cluster_method Hierarchical clustering method
--aligners "star" OR "bwa" OR "tophat2" OR "hisat2" OR "none","none" for bam files
--expression_analysis_mode "DESeq2" OR "edgeR" OR "none"
--peakCalling_mode "group" OR "independence" for MATK and MeTPeak
--peakMerged_mode "rank" OR "macs2" OR "MATK" OR "metpeak" OR "mspc"
--methylation_analysis_mode "MATK" OR "QNB" OR "Wilcox-test" OR "edgeR" OR "DESeq2"
Process skipping options:
--skip_fastp Skip fastp
--skip_fastqc Skip FastQC
--skip_rseqc Skip RSeQC
--skip_filterrRNA Skip the process of rRNA cleaning before mapping
--skip_qc Skip all Quality Control steps
--skip_edger Skip the EdgeR process of differential expression analysis steps
--skip_deseq2 Skip the DESeq2 process of differential expression analysis steps
--skip_sort Skip the process of sorting BAM files
--skip_expression Skip all Differential expression analysis
--skip_peakCalling Skip all Peak Calling steps
--skip_diffpeakCalling Skip all Differential methylation analysis
--skip_metpeak Skip the MeTPeak process of Peak Calling steps
--skip_macs2 Skip the MACS2 process of Peak Calling steps
--skip_matk Skip the MATK process of Peak Calling steps
--skip_meyer Skip the Meyer process of Peak Calling steps
--skip_QNB Skip the QNB process of Differential methylation analysis
--skip_diffmatk Skip the MATK process of Differential methylation analysis
--skip_annotation Skip the process of peak annotation
--skip_motif Skip the process of motif searching
--skip_m6Aprediction Skip the process of m6A site prediction
--skip_createbedgraph Skip the process of making bedgraph format files and creatIGVjs
Other options:
--outdir The output directory where the results will be saved, defalut = $baseDir/results
--help To show the help message
-name Name for the pipeline run. If not specified, Nextflow will automatically generate a random mnemonic.
""".stripIndent()
}
// Show help message
if (params.help) {
helpMessage()
exit 0
}
// Stage config files
ch_multiqc_config = file(params.multiqc_config, checkIfExists: true)
ch_output_docs = file("$baseDir/docs/output.md", checkIfExists: true)
/*
* SET UP CONFIGURATION VARIABLES
*/
// Configurable variables
params.name = false
params.project = false
params.genome = false
params.call = false
params.email = false
params.plaintext_email = false
params.seqCenter = false
params.help = false
// Validate inputs
// 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(", ")}"
}
params.star_index = params.genome ? params.genomes[ params.genome ].star ?: false : false
params.fasta = params.genome ? params.genomes[ params.genome ].fasta ?: false : false
params.gtf = params.genome ? params.genomes[ params.genome ].gtf ?: false : false
params.bed12 = params.genome ? params.genomes[ params.genome ].bed12 ?: false : false
params.hisat2_index = params.genome ? params.genomes[ params.genome ].hisat2 ?: false : false
// 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
}
//check fasta files
if ( params.fasta ){
fasta = file(params.fasta, checkIfExists: true)
if( !fasta.exists() ) exit 1, LikeletUtils.print_red("Fasta file not found: ${params.fasta}")
}else {
exit 1, LikeletUtils.print_red("No reference genome specified!")
}
//check gtf files
if( params.gtf ){
gtf = file ( params.gtf, checkIfExists: true)
if( !gtf.exists() ) exit 1, LikeletUtils.print_red("gtf not found: ${params.gtf}")
} else {
exit 1, LikeletUtils.print_red("No GTF annotation specified!")
}
//check rRNA fasta files
if ( params.rRNA_fasta ){
rRNA_fasta = file(params.rRNA_fasta, checkIfExists: true)
if( !rRNA_fasta.exists() ) exit 1, LikeletUtils.print_red("rRNA fasta file not found: ${params.rRNA_fasta}")
}else{
rRNA_fasta = Channel.from("")
}
//check comparefile
if( params.comparefile == "two_groups" ){
comparefile = false
compareLines = Channel.from("two_groups")
}else if( params.comparefile){
comparefile = file(params.comparefile)
if( !comparefile.exists() ) exit 1, print_red("Compare file not found: ${params.comparefile}")
compareLines = Channel.from(comparefile.readLines())
}else {
comparefile = false
compareLines = Channel.from("")
}
compareLines.into{
compareLines_for_DESeq2; compareLines_for_edgeR; compareLines_for_plot;
compareLines_for_diffm6A; compareLines_for_arranged_result
}
// Validate the params of skipping Aligners Tools Setting
if( params.aligners == "none" || params.aligners == "star" || params.aligners == "hisat2" || params.aligners == "tophat2" || params.aligners == "bwa" ){
aligner = params.aligners
}else{
exit 1, LikeletUtils.print_red("Invalid aligner option: ${params.aligner}. Valid options: 'star', 'hisat2', 'tophat2', 'bwa', 'none'")
}
if( params.expression_analysis_mode == "edgeR" ){
params.skip_edger = false
params.skip_deseq2 = true
params.skip_expression = false
}else if( params.expression_analysis_mode == "DESeq2" ){
params.skip_edger = true
params.skip_deseq2 = false
params.skip_expression = false
}else if( params.expression_analysis_mode == "none" ){
params.skip_edger = true
params.skip_deseq2 = true
params.skip_expression = true
}else{
exit 1, LikeletUtils.print_red("Invalid expression_analysis_mode option: ${params.expression_analysis_mode}. Valid options: 'edgeR', 'DESeq2', 'none'")
}
/*
* Create a channel for input read files
*/
if ( params.readPaths ){
if (aligner == 'none') {
Channel
.fromPath( params.readPaths )
.ifEmpty { exit 1, "params.readPaths was empty - no input files supplied" }
.into{ raw_fastq; raw_bam }
} else if ( params.single_end ) {
Channel
.from( params.readPaths )
.map{ row -> [ row[0], [ file(row[1][0], checkIfExists: true) ] ] }
.ifEmpty { exit 1, "params.readPaths was empty - no input files supplied" }
.into{ raw_fastq; raw_bam }
} else {
Channel
.from( params.readPaths )
.map{ row -> [ row[0], [ file(row[1][0], checkIfExists: true), file(row[1][1], checkIfExists: true) ] ] }
.ifEmpty { exit 1, "params.readPaths was empty - no input files supplied" }
.into{ raw_fastq; raw_bam }
}
}else if( params.designfile ) {
designfile = file(params.designfile, checkIfExists: true)
if( !designfile.exists() ) exit 1, LikeletUtils.print_red("Design file not found: ${params.designfile}")
LikeletUtils.extractData(designfile).into{ input_data_fastq; input_data_bam; designinfo}
input_data_fastq.filter{ it[6] == "fastq" }.map{ it.subList(0,6) }.set{ raw_fastq } //filter filetype & delete filetype
input_data_bam.filter{ it[6] == "bam" }.map{ it.subList(0,6) }.set{ raw_bam }
designinfo.map{ it.getAt([0,5]) }.set{ format_design }
}else{
exit 1, LikeletUtils.print_red("No Design file specified!")
}
/*
showing the process and files
========================================================================================
*/
// Header log info
println LikeletUtils.sysucc_ascii()
println LikeletUtils.print_purple("============You are running MeRIPseqPipe with the following parameters===============")
println LikeletUtils.print_purple("Checking parameters ...")
println LikeletUtils.print_yellow("===================================Pipeline summary=============================")
println (LikeletUtils.print_yellow("Max Resources : ") + LikeletUtils.print_green("$params.max_memory memory, $params.max_cpus cpus, $params.max_time time per job"))
if (workflow.containerEngine){
println (LikeletUtils.print_yellow("Container : ") + LikeletUtils.print_green("$workflow.containerEngine - $workflow.container"))
}
println (LikeletUtils.print_yellow("Output dir : ") + LikeletUtils.print_green(params.outdir))
println (LikeletUtils.print_yellow("Launch dir : ") + LikeletUtils.print_green(workflow.launchDir))
println (LikeletUtils.print_yellow("Working dir : ") + LikeletUtils.print_green(workflow.workDir))
println (LikeletUtils.print_yellow("Script dir : ") + LikeletUtils.print_green(workflow.projectDir))
println (LikeletUtils.print_yellow("User : ") + LikeletUtils.print_green(workflow.userName))
if (workflow.profile == 'awsbatch') {
println (LikeletUtils.print_yellow("AWS Region : ") + LikeletUtils.print_green(params.awsregion))
println (LikeletUtils.print_yellow("AWS Queue : ") + LikeletUtils.print_green(params.awsqueue))
}
println (LikeletUtils.print_yellow("Config Profile : ") + LikeletUtils.print_green(workflow.profile))
if (params.config_profile_description){
println (LikeletUtils.print_yellow("Config Description : ") + LikeletUtils.print_green(params.config_profile_description))
}
if (params.config_profile_contact){
println (LikeletUtils.print_yellow("Config Contact : ") + LikeletUtils.print_green(params.config_profile_contact))
}
if (params.config_profile_url){
println (LikeletUtils.print_yellow("Config URL : ") + LikeletUtils.print_green(params.config_profile_url))
}
if (params.email || params.email_on_fail) {
println (LikeletUtils.print_yellow("E-mail Address : ") + LikeletUtils.print_green(params.email))
println (LikeletUtils.print_yellow("E-mail on failure : ") + LikeletUtils.print_green(params.email_on_fail))
println (LikeletUtils.print_yellow("MultiQC maxsize : ") + LikeletUtils.print_green(params.maxMultiqcEmailFileSize))
}
println LikeletUtils.print_yellow("=====================================Reads types================================")
println (LikeletUtils.print_yellow("SingleEnd : ") + LikeletUtils.print_green(params.single_end ? 'Single-End' : 'Paired-End'))
println (LikeletUtils.print_yellow("Stranded : ") + LikeletUtils.print_green(params.stranded))
println (LikeletUtils.print_yellow("gzip : ") + LikeletUtils.print_green(params.gzip))
println LikeletUtils.print_yellow("====================================Mode selected==============================")
println (LikeletUtils.print_yellow("aligners : ") + LikeletUtils.print_green(params.aligners))
println (LikeletUtils.print_yellow("peakCalling_mode : ") + LikeletUtils.print_green(params.peakCalling_mode))
println (LikeletUtils.print_yellow("peakMerged_mode : ") + LikeletUtils.print_green(params.peakMerged_mode))
println (LikeletUtils.print_yellow("expression_analysis_mode : ") + LikeletUtils.print_green(params.expression_analysis_mode))
println (LikeletUtils.print_yellow("methylation_analysis_mode : ") + LikeletUtils.print_green(params.methylation_analysis_mode))
println LikeletUtils.print_yellow("==================================Input files selected==========================")
println (LikeletUtils.print_yellow("Reads Path : ") + LikeletUtils.print_green(params.readPaths ? "github" : params.input))
println (LikeletUtils.print_yellow("fasta file : ") + LikeletUtils.print_green(params.fasta))
println (LikeletUtils.print_yellow("Gtf file : ") + LikeletUtils.print_green(params.gtf))
println (LikeletUtils.print_yellow("Design file : ") + LikeletUtils.print_green(params.designfile))
println (LikeletUtils.print_yellow("Compare file : ") + LikeletUtils.print_green(params.comparefile))
println LikeletUtils.print_yellow("==================================Skip model selected==========================")
println (LikeletUtils.print_yellow("Skip samtools sort : ") + LikeletUtils.print_green(params.skip_sort))
println (LikeletUtils.print_yellow("Skip expression analysis : ") + LikeletUtils.print_green(params.skip_expression))
println (LikeletUtils.print_yellow("Skip peakCalling : ") + LikeletUtils.print_green(params.skip_peakCalling))
println (LikeletUtils.print_yellow("Skip diffpeakCalling : ") + LikeletUtils.print_green(params.skip_diffpeakCalling))
println (LikeletUtils.print_yellow("Skip annotation : ") + LikeletUtils.print_green(params.skip_annotation))
println (LikeletUtils.print_yellow("Skip qc : ") + LikeletUtils.print_green(params.skip_qc))
// log.info summary.collect { k,v -> "${k.padRight(18)}: $v" }.join("\n")
// // Check the hostnames against configured profiles
// checkHostname()
/*
========================================================================================
check or build the index
========================================================================================
*/
/*
* PREPROCESSING - Build BED12 file
* NEED gtf.file
*/
process CheckDesignCompare{
input:
val design_info from format_design.collect()
file comparefile
output:
file (formatted_design) into formatted_designfile
script:
formatted_design = "formatted_designfile.txt"
formatted_design_info = ""
for(int i = 0; i < design_info.size(); i+=2 ) {
sample = design_info[i] + ".input," + design_info[i] + ".ip"
formatted_design_info += design_info[i] + "," + sample + "," + design_info[i+1] + "\n"
}
"""
echo "Sample_ID,input_FileName,ip_FileName,Group" > $formatted_design
echo "$formatted_design_info" |awk NF |sort | uniq >> $formatted_design
# Check the consistency of designfile and comparefile
if [ "$comparefile" != "input.1" ]; then
## get groups' name in comparefile
cat $comparefile | dos2unix | awk -F "_vs_" '{print \$1"\\n"\$2}' | sort | uniq > tmp.compare.group
## get groups' name in designfile
awk -F, 'NR>1{print \$4}' $formatted_design | sort | uniq > tmp.design.group
intersection_num=\$(join tmp.compare.group tmp.design.group | wc -l)
if [[ \$intersection_num != \$(cat tmp.compare.group| wc -l) ]] ;then
echo "The groups' name of comparefile and designfile are inconsistent."
echo "Please check your comparefile: "$comparefile
echo "The groups' name of comparefile in designfile: "\$(join tmp.compare.group tmp.design.group)
exit 1
fi
rm tmp.compare.group tmp.design.group
fi
"""
}
process makeBED12 {
label 'build_index'
tag "gtf2bed12"
publishDir path: { params.saveReference ? "${params.outdir}/Genome/reference_genome" : params.outdir },
saveAs: { params.saveReference ? it : null }, mode: 'copy'
when:
!params.skip_qc && !params.skip_rseqc || !params.skip_motif
input:
file gtf
output:
file "${gtf.baseName}.bed" into bed12file
shell:
'''
gtf_file=!{gtf}
gtfToGenePred -genePredExt -geneNameAsName2 $gtf_file ${gtf_file/.gtf/.tmp}
genePredToBed ${gtf_file/.gtf/.tmp} ${gtf_file/.gtf/.bed}
'''
}
process makechromesize {
label 'build_index'
tag "gtf2bed12"
publishDir path: { params.saveReference ? "${params.outdir}/Genome/reference_genome" : params.outdir },
saveAs: { params.saveReference ? it : null }, mode: 'copy'
when:
true
input:
file fasta
output:
file "chromsizes.file" into chromsizesfile
shell:
"""
samtools faidx ${fasta}
cut -f1,2 ${fasta}.fai > chromsizes.file
"""
}
/*
* PREPROCESSING - Build TOPHAT2 index
* NEED genome.fa
*/
if( params.tophat2_index && aligner == "tophat2" ){
tophat2_index = Channel
.fromPath( params.tophat2_index )
.ifEmpty { exit 1, "Tophat2 index not found: ${params.tophat2_index}" }
}else if( params.fasta ){
process MakeTophat2Index {
label 'build_index'
tag "tophat2_index"
publishDir path: { params.saveReference ? "${params.outdir}/Genome/": params.outdir },
saveAs: { params.saveReference ? it : null }, mode: 'copy'
input:
file fasta
output:
file "Tophat2Index/*" into tophat2_index
when:
aligner == "tophat2"
script:
tophat2_index = "Tophat2Index/" + fasta.baseName.toString()
"""
mkdir Tophat2Index
ln $fasta Tophat2Index
bowtie2-build -f $fasta $tophat2_index
"""
}
}else {
exit 1, println LikeletUtils.print_red("There is no Tophat2 Index")
}
/*
* PREPROCESSING - Build HISAT2 index
* NEED genome.fa genes.gtf snp.txt/vcf
*/
if( params.hisat2_index && aligner == "hisat2" ){
hisat2_index = Channel
.fromPath(params.hisat2_index)
.ifEmpty { exit 1, "hisat2 index not found: ${params.hisat2_index}" }
}else if( params.fasta){
process MakeHisat2Index {
label 'build_index'
tag "hisat2_index"
publishDir path: { params.saveReference ? "${params.outdir}/Genome/ " : params.outdir },
saveAs: { params.saveReference ? it : null }, mode: 'copy'
input:
file fasta
file gtf
output:
file "Hisat2Index/*" into hisat2_index
when:
aligner == "hisat2"
script:
"""
mkdir Hisat2Index
hisat2_extract_exons.py $gtf > Hisat2Index/${fasta.baseName}.exon
hisat2_extract_splice_sites.py $gtf > Hisat2Index/${fasta.baseName}.ss
hisat2-build -p ${task.cpus} -f $fasta --exon Hisat2Index/${fasta.baseName}.exon --ss Hisat2Index/${fasta.baseName}.ss Hisat2Index/${fasta.baseName}
"""
}
}else {
exit 1, println LikeletUtils.print_red("There is no Hisat2 Index")
}
/*
* PREPROCESSING - Build BWA index
* NEED genome.fa
*/
if( params.bwa_index && aligner == "bwa" ){
bwa_index = Channel
.fromPath( params.bwa_index )
.ifEmpty { exit 1, "bwa index not found: ${params.bwa_index}" }
}else if(params.fasta ){
process MakeBWAIndex {
label 'build_index'
tag "bwa_index"
publishDir path: { params.saveReference ? "${params.outdir}/Genome/" : params.outdir },
saveAs: { params.saveReference ? it : null }, mode: 'copy'
input:
file fasta
output:
file "BWAIndex/*" into bwa_index
when:
aligner == "bwa"
script:
"""
mkdir BWAIndex
cd BWAIndex/
bwa index -p ${fasta.baseName} -a bwtsw ../$fasta
cd ../
"""
}
}else {
exit 1, println LikeletUtils.print_red("There is no BWA Index")
}
/*
* PREPROCESSING - Build STAR index
* NEED genome.fa genes.gtf
*/
if( params.star_index && aligner == "star"){
star_index = Channel
.fromPath(params.star_index)
.ifEmpty { exit 1, "STAR index not found: ${params.star_index}" }
}else if( params.fasta ){
process MakeStarIndex {
label 'build_index'
tag "star_index"
publishDir path: { params.saveReference ? "${params.outdir}/Genome/" : params.outdir },
saveAs: { params.saveReference ? it : null }, mode: 'copy'
input:
file fasta
file gtf
output:
file "StarIndex" into star_index
when:
aligner == "star"
script:
readLength = 50
overhang = readLength - 1
"""
mkdir StarIndex
STAR --runThreadN ${task.cpus} \
--runMode genomeGenerate \
--genomeDir StarIndex \
--genomeFastaFiles $fasta \
--sjdbGTFfile $gtf \
--sjdbOverhang $overhang \
--limitGenomeGenerateRAM 36000000000
"""
}
}else {
exit 1, println LikeletUtils.print_red("There is no STAR Index")
}
/*
* PREPROCESSING - Build rRNA index
* NEED rRNA.fa
*/
process MakerRNAindex {
label 'build_index'
tag "rRNA_index"
publishDir path: { params.saveReference ? "${params.outdir}/Genome/" : params.outdir },
saveAs: { params.saveReference ? it : null }, mode: 'copy'
input:
file rRNA_fasta from rRNA_fasta
output:
file "rRNAindex/*" into rRNA_index
when:
params.rRNA_fasta && !params.skip_filterrRNA
script:
"""
mkdir rRNAindex
hisat2-build -p ${task.cpus} -f $rRNA_fasta rRNAindex/${rRNA_fasta.baseName}
"""
}
/*
========================================================================================
Step 1. QC------Fastp & FastQC
========================================================================================
*/
process Fastp{
label 'fastp'
tag "$sample_name"
//errorStrategy 'ignore'
publishDir path: { params.skip_fastp ? params.outdir : "${params.outdir}/QC/fastp" },
saveAs: { params.skip_fastp ? null : it }, mode: 'link'
input:
set val(sample_id), file(reads), val(reads_single_end), val(gzip), val(input), val(group) from raw_fastq
output:
set val(sample_name), file("*_aligners.fastq*"), val(reads_single_end), val(sample_id), val(gzip), val(input), val(group) into fastqc_reads, fastp_reads
file "*" into fastp_results
when:
aligner != "none"
shell:
skip_fastp = params.skip_fastp
if ( reads_single_end ){
filename = reads.toString() - ~/(\.fq)?(\.fastq)?(\.gz)?$/
sample_name = filename
add_aligners = sample_name + "_aligners.fastq" + (gzip ? ".gz" : "")
"""
if [ $skip_fastp == "false" ]; then
fastp -i ${reads} -o ${add_aligners} -j ${sample_name}_fastp.json -h ${sample_name}_fastp.html -w ${task.cpus}
else
mv ${reads} ${add_aligners}
fi
"""
} else {
filename = reads[0].toString() - ~/(_R[0-9])?(_[0-9])?(\.fq)?(\.fastq)?(\.gz)?$/
sample_name = filename
add_aligners_1 = sample_name + "_1_aligners.fastq" + (gzip ? ".gz" : "")
add_aligners_2 = sample_name + "_2_aligners.fastq" + (gzip ? ".gz" : "")
"""
if [ $skip_fastp == "false" ]; then
fastp -i ${reads[0]} -o ${add_aligners_1} -I ${reads[1]} -O ${add_aligners_2} -j ${sample_name}_fastp.json -h ${sample_name}_fastp.html -w ${task.cpus}
else
mv ${reads[0]} ${add_aligners_1}
mv ${reads[1]} ${add_aligners_2}
fi
"""
}
}
process Fastqc{
tag "$sample_name"
publishDir path: { params.skip_fastqc ? params.outdir : "${params.outdir}/QC" },
saveAs: { params.skip_fastqc ? null : it }, mode: 'link'
input:
set val(sample_name), file(reads), val(reads_single_end), val(sample_id), val(gzip), val(input), val(group) from fastqc_reads
output:
file "fastqc/*" into fastqc_results
when:
aligner != "none" && !params.skip_fastqc
shell:
skip_fastqc = params.skip_fastqc
if ( reads_single_end){
"""
mkdir fastqc
fastqc -o fastqc --noextract ${reads}
"""
} else {
"""
mkdir fastqc
fastqc -o fastqc --noextract ${reads[0]}
fastqc -o fastqc --noextract ${reads[1]}
"""
}
}
/*
========================================================================================
Step 2. Reads Mapping
========================================================================================
*/
if(params.rRNA_fasta && !params.skip_filterrRNA){
fastp_reads.set{rRNA_reads}
process FilterrRNA {
label 'aligners'
tag "$sample_name"
publishDir "${params.outdir}/alignment/rRNA_dup", mode: 'link', overwrite: true
input:
set val(sample_name), file(reads), val(reads_single_end), val(sample_id), val(gzip), val(input), val(group) from rRNA_reads
file index from rRNA_index.collect()
output:
set val(sample_name), file("*.fastq.gz"), val(reads_single_end), val(sample_id), val(gzip), val(input), val(group) into tophat2_reads, hisat2_reads, bwa_reads, star_reads
file "*_summary.txt" into rRNA_log
when:
params.rRNA_fasta && !params.skip_filterrRNA
script:
gzip = true
index_base = index[0].toString() - ~/(\.exon)?(\.\d)?(\.fa)?(\.gtf)?(\.ht2)?$/
if (reads_single_end) {
"""
hisat2 --summary-file ${sample_name}_rRNA_summary.txt \
--no-spliced-alignment --no-softclip --norc --no-unal \
-p ${task.cpus} --dta --un-gz ${sample_id}.fastq.gz \
-x $index_base \
-U $reads | \
samtools view -@ ${task.cpus} -Shub - | \
samtools sort -@ ${task.cpus} -o ${sample_name}_rRNA_sort.bam -
"""
} else {
"""
hisat2 --summary-file ${sample_name}_rRNA_summary.txt \
--no-spliced-alignment --no-softclip --norc --no-unal \
-p ${task.cpus} --dta --un-conc-gz ${sample_name}_fastq.gz \
-x $index_base \
-1 ${reads[0]} -2 ${reads[1]} | \
samtools view -@ ${task.cpus} -Shub - | \
samtools sort -@ ${task.cpus} -o ${sample_name}_rRNA_sort.bam -
mv ${sample_name}_fastq.1.gz ${sample_name}_1.fastq.gz
mv ${sample_name}_fastq.2.gz ${sample_name}_2.fastq.gz
"""
}
}
}else{
fastp_reads.into{tophat2_reads; hisat2_reads; bwa_reads; star_reads}
}
process Tophat2Align {
label 'aligners'
tag "$sample_name"
publishDir "${params.outdir}/alignment/tophat2", mode: 'link', overwrite: true
input:
set val(sample_name), file(reads), val(reads_single_end), val(sample_id), val(gzip), val(input), val(group) from tophat2_reads
file index from tophat2_index.collect()
file gtf
output:
set val(sample_id), file("*_tophat2.bam"), val(reads_single_end), val(gzip), val(input), val(group) into tophat2_bam
file "*_log.txt" into tophat2_log
when:
aligner == "tophat2"
script:
index_base = index[0].toString() - ~/(\.rev)?(\.\d)?(\.fa)?(\.bt2)?$/
strand_info = params.stranded == "no" ? "fr-unstranded" : params.stranded == "reverse" ? "fr-secondstrand" : "fr-firststrand"
if (reads_single_end) {
"""
tophat -p ${task.cpus} \
-G $gtf \
-o $sample_name \
--no-novel-juncs \
--library-type $strand_info \
$index_base \
$reads > ${sample_name}_log.txt
mv $sample_name/accepted_hits.bam ${sample_name}_tophat2.bam
"""
} else {
"""
tophat -p ${task.cpus} \
-G $gtf \
-o $sample_name \
--no-novel-juncs \
--library-type $strand_info \
$index_base \
${reads[0]} ${reads[1]} > ${sample_name}_log.txt
mv $sample_name/accepted_hits.bam ${sample_name}_tophat2.bam
"""
}
}
process Hisat2Align {
label 'aligners'
tag "$sample_name"
publishDir "${params.outdir}/alignment/hisat2", mode: 'link', overwrite: true
input:
set val(sample_name), file(reads), val(reads_single_end), val(sample_id), val(gzip), val(input), val(group) from hisat2_reads
file index from hisat2_index.collect()
output:
set val(sample_id), file("*_hisat2.bam"), val(reads_single_end), val(gzip), val(input), val(group) into hisat2_bam
file "*_summary.txt" into hisat2_log
when:
aligner == "hisat2"
script:
index_base = index[0].toString() - ~/(\.exon)?(\.\d)?(\.fa)?(\.gtf)?(\.ht2)?$/
if (reads_single_end) {
"""
hisat2 --summary-file ${sample_name}_hisat2_summary.txt\
-p ${task.cpus} --dta \
-x $index_base \
-U $reads | \
samtools view -@ ${task.cpus} -hbS - > ${sample_name}_hisat2.bam
"""
} else {
"""
hisat2 --summary-file ${sample_name}_hisat2_summary.txt \
-p ${task.cpus} --dta \
-x $index_base \
-1 ${reads[0]} -2 ${reads[1]} | \
samtools view -@ ${task.cpus} -hbS - > ${sample_name}_hisat2.bam
"""
}
}
process BWAAlign{
label 'aligners'
tag "$sample_name"
publishDir "${params.outdir}/alignment/bwa", mode: 'link', overwrite: true
input:
set val(sample_name), file(reads), val(reads_single_end), val(sample_id), val(gzip), val(input), val(group) from bwa_reads
file index from bwa_index.collect()
output:
set val(sample_id), file("*_bwa.bam"), val(reads_single_end), val(gzip), val(input), val(group) into bwa_bam
file "*" into bwa_result
when:
aligner == "bwa"
script:
index_base = index[0].toString() - ~/(\.pac)?(\.bwt)?(\.ann)?(\.amb)?(\.sa)?(\.fa)?$/
if (reads_single_end) {
"""
bwa aln -t ${task.cpus} \
-f ${reads.baseName}.sai \
$index_base \
$reads
bwa samse \
$index_base \
${reads.baseName}.sai \
$reads | \
samtools view -@ ${task.cpus} -h -bS - > ${sample_name}_bwa.bam
"""
} else {
"""
bwa aln -t ${task.cpus} \
-f ${reads[0].baseName}.sai \
$index_base \
${reads[0]}
bwa aln -t ${task.cpus} \
-f ${reads[1].baseName}.sai \
$index_base \
${reads[1]}
bwa sampe \
$index_base \
${reads[0].baseName}.sai ${reads[1].baseName}.sai \
${reads[0]} ${reads[1]} | \
samtools view -@ ${task.cpus} -h -bS - > ${sample_name}_bwa.bam
"""
}
}
process StarAlign {
label 'aligners'
tag "$sample_name"
publishDir "${params.outdir}/alignment/star", mode: 'link', overwrite: true
input:
set val(sample_name), file(reads), val(reads_single_end), val(sample_id), val(gzip), val(input), val(group) from star_reads
file star_index from star_index.collect()
output:
set val(sample_id), file("*_star.bam"), val(reads_single_end), val(gzip), val(input), val(group) into star_bam
file "*.final.out" into star_log
when:
aligner == "star"
script:
gzip_cmd = gzip ? "--readFilesCommand zcat" : ""
if (reads_single_end) {
"""
STAR --runThreadN ${task.cpus} $gzip_cmd \
--twopassMode Basic \
--genomeDir $star_index \
--readFilesIn $reads \
--outSAMtype BAM Unsorted \
--alignSJoverhangMin 8 --alignSJDBoverhangMin 1 \
--outFilterIntronMotifs RemoveNoncanonical \
--outFilterMultimapNmax 20 \
--alignIntronMin 20 \
--alignIntronMax 100000 \
--alignMatesGapMax 1000000 \
--outFileNamePrefix ${sample_name} > ${sample_name}_log.txt
mv ${sample_name}Aligned.out.bam ${sample_name}_star.bam
"""
} else {
"""
STAR --runThreadN ${task.cpus} $gzip_cmd \
--twopassMode Basic \
--genomeDir $star_index \
--readFilesIn ${reads[0]} ${reads[1]} \
--outSAMtype BAM Unsorted \
--alignSJoverhangMin 8 --alignSJDBoverhangMin 1 \
--outFilterIntronMotifs RemoveNoncanonical \
--outFilterMultimapNmax 20 \
--alignIntronMin 20 \
--alignIntronMax 1000000 \
--alignMatesGapMax 1000000 \
--outFileNamePrefix ${sample_name} > ${sample_name}_log.txt
mv ${sample_name}Aligned.out.bam ${sample_name}_star.bam
"""
}
}
/*
========================================================================================
Step 3 Sort BAM file AND QC
========================================================================================
*/
Channel
.from()
.concat(tophat2_bam, hisat2_bam, bwa_bam, star_bam, raw_bam)
.set{ merge_bam_file }
/*
* STEP 3-1 - Sort BAM file
*/
process SortRename {
label 'sort'
tag "$sample_name"
publishDir "${params.outdir}/alignment/samtoolsSort/", mode: 'link', overwrite: true
input:
set val(sample_id), file(bam_file), val(reads_single_end), val(gzip), val(input), val(group) from merge_bam_file
output:
set val(group), val(sample_id), file("*.bam"), file("*.bai") into sorted_bam
file "*.{bam,bai}" into rseqc_bam, bedgraph_bam, feacount_bam, cuffbam, peakquan_bam, diffpeak_bam, sng_bam
file "*.bam" into bam_results
script:
sample_name = sample_id + (input ? ".input_" : ".ip_") + group
output = sample_name + ".bam"
mapq_cutoff = (params.mapq_cutoff).toInteger()
if (!params.skip_sort){
"""
if [ "$mapq_cutoff" -gt "0" ]; then
samtools view -hbq $mapq_cutoff $bam_file | samtools sort -@ ${task.cpus} -O BAM -o $output -
else
samtools sort -@ ${task.cpus} -O BAM -o $output $bam_file
fi
samtools index -@ ${task.cpus} $output
"""
} else {
"""
if [ "$mapq_cutoff" -gt "0" ]; then
samtools view -hbq $mapq_cutoff $bam_file > $output
else
mv $bam_file $output
fi
samtools index -@ ${task.cpus} $output
"""
}
}
/*
* STEP 3-2 - RSeQC analysis
*/
process RSeQC {
tag "$output"
publishDir "${params.outdir}/QC/rseqc" , mode: 'link', overwrite: true,
saveAs: {filename ->
if (filename.indexOf("bam_stat.txt") > 0) "bam_stat/$filename"
else if (filename.indexOf("infer_experiment.txt") > 0) "infer_experiment/$filename"
else if (filename.indexOf("read_distribution.txt") > 0) "read_distribution/$filename"
else if (filename.indexOf("read_duplication.DupRate_plot.pdf") > 0) "read_duplication/$filename"
else if (filename.indexOf("read_duplication.DupRate_plot.r") > 0) "read_duplication/rscripts/$filename"
else if (filename.indexOf("read_duplication.pos.DupRate.xls") > 0) "read_duplication/dup_pos/$filename"
else if (filename.indexOf("read_duplication.seq.DupRate.xls") > 0) "read_duplication/dup_seq/$filename"
else if (filename.indexOf("inner_distance.txt") > 0) "inner_distance/$filename"
else if (filename.indexOf("inner_distance_freq.txt") > 0) "inner_distance/data/$filename"
else if (filename.indexOf("inner_distance_plot.r") > 0) "inner_distance/rscripts/$filename"
else if (filename.indexOf("inner_distance_plot.pdf") > 0) "inner_distance/plots/$filename"
else if (filename.indexOf("junction_plot.r") > 0) "junction_annotation/rscripts/$filename"
else if (filename.indexOf("junction.xls") > 0) "junction_annotation/data/$filename"
else if (filename.indexOf("splice_events.pdf") > 0) "junction_annotation/events/$filename"
else if (filename.indexOf("splice_junction.pdf") > 0) "junction_annotation/junctions/$filename"
else if (filename.indexOf("junctionSaturation_plot.pdf") > 0) "junction_saturation/$filename"
else if (filename.indexOf("junctionSaturation_plot.r") > 0) "junction_saturation/rscripts/$filename"
else filename
}
when:
!params.skip_qc && !params.skip_rseqc
input:
file bam_file from rseqc_bam
file bed12 from bed12file.collect()
output:
file "*" into rseqc_results
file "*.bam_stat.txt" into bam_stat_for_normlization
script:
output = bam_file[0].toString() - ~/(\.bam)?$/
"""
infer_experiment.py -i ${bam_file[0]} -r ${bed12} > ${output}.infer_experiment.txt
junction_annotation.py -i ${bam_file[0]} -o ${output}.rseqc -r ${bed12}
bam_stat.py -i ${bam_file[0]} > ${output}.bam_stat.txt
junction_saturation.py -i ${bam_file[0]} -o ${output}.rseqc -r ${bed12} 2> ${output}.junction_annotation_log.txt