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main.nf
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#!/usr/bin/env nextflow
/*
========================================================================================
nf-core/nanoseq
========================================================================================
nf-core/nanoseq Analysis Pipeline.
#### Homepage / Documentation
https://github.com/nf-core/nanoseq
----------------------------------------------------------------------------------------
*/
def helpMessage() {
log.info nfcoreHeader()
log.info"""
Usage:
The typical command for running the pipeline is as follows:
nextflow run nf-core/nanoseq \
--input samplesheet.csv \
--protocol DNA \
--input_path ./fast5/ \
--flowcell FLO-MIN106 \
--kit SQK-LSK109 \
--barcode_kit SQK-PBK004 \
-profile docker
Mandatory arguments
--input [file] Comma-separated file containing information about the samples in the experiment (see docs/usage.md)
--protocol [str] Specifies the type of sequencing i.e. "DNA", "cDNA" or "directRNA"
-profile [str] Configuration profile to use. Can use multiple (comma separated)
Available: docker, singularity, awsbatch, test and more.
Basecalling/Demultiplexing
--input_path [file] Path to Nanopore run directory (e.g. fastq_pass/) or a basecalled fastq file that requires demultiplexing
--flowcell [str] Flowcell used to perform the sequencing e.g. FLO-MIN106. Not required if '--guppy_config' is specified
--kit [str] Kit used to perform the sequencing e.g. SQK-LSK109. Not required if '--guppy_config' is specified
--barcode_kit [str] Barcode kit used to perform the sequencing e.g. SQK-PBK004
--guppy_config [file/str] Guppy config file used for basecalling. Cannot be used in conjunction with '--flowcell' and '--kit'
--guppy_model [file/str] Custom basecalling model file (JSON) to use for Guppy basecalling, such as the output from Taiyaki (Default: false)
--guppy_gpu [bool] Whether to perform basecalling with Guppy in GPU mode (Default: false)
--guppy_gpu_runners [int] Number of '--gpu_runners_per_device' used for Guppy when using '--guppy_gpu' (Default: 6)
--guppy_cpu_threads [int] Number of '--cpu_threads_per_caller' used for Guppy when using '--guppy_gpu' (Default: 1)
--gpu_device [str] Basecalling device specified to Guppy in GPU mode using '--device' (Default: 'auto')
--gpu_cluster_options [str] Cluster options required to use GPU resources (e.g. '--part=gpu --gres=gpu:1')
--qcat_min_score [int] Minimum scores of '--min-score' used for qcat (Default: 60)
--qcat_detect_middle [bool] Search for adapters in the whole read '--detect-middle' used for qcat (Default: false)
--skip_basecalling [bool] Skip basecalling with Guppy (Default: false)
--skip_demultiplexing [bool] Skip demultiplexing with Guppy (Default: false)
Alignment
--aligner [str] Specifies the aligner to use (available are: minimap2 or graphmap2). Both are capable of performing unspliced/spliced alignment (Default: 'minimap2')
--stranded [bool] Specifies if the data is strand-specific. Automatically activated when using '--protocol directRNA' (Default: false)
--save_align_intermeds [bool] Save the '.sam' files from the alignment step (Default: false)
--skip_alignment [bool] Skip alignment and subsequent process (Default: false)
Coverage tracks
--skip_bigwig [bool] Skip BigWig file generation (Default: false)
--skip_bigbed [bool] Skip BigBed file generation (Default: false)
QC
--skip_qc [bool] Skip all QC steps apart from MultiQC (Default: false)
--skip_pycoqc [bool] Skip pycoQC (Default: false)
--skip_nanoplot [bool] Skip NanoPlot (Default: false)
--skip_fastqc [bool] Skip FastQC (Default: false)
--skip_multiqc [bool] Skip MultiQC (Default: false)
Transcript Quantification
--transcriptquant [str] Specifies the transcript quantification method to use (available are: bambu or stringtie2). Only available when protocol is cDNA or directRNA.
--skip_transcriptquant [bool] Skip transcript quantification and subsequent process (Default: false)
Other
--outdir [file] The output directory where the results will be saved (Default: '/results')
--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 (Default: false)
--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
--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 (Default: 'eu-west-1')
--awscli [str] Path to the AWS CLI tool
""".stripIndent()
}
// Show help message
if (params.help) {
helpMessage()
exit 0
}
/*
* SET UP CONFIGURATION VARIABLES
*/
if (params.input) { ch_input = file(params.input, checkIfExists: true) } else { exit 1, "Samplesheet file not specified!" }
// Function to check if running offline
def isOffline() {
try {
return NXF_OFFLINE as Boolean
}
catch( Exception e ) {
return false
}
}
def ch_guppy_model = Channel.empty()
def ch_guppy_config = Channel.empty()
if (!params.skip_basecalling) {
// Pre-download test-dataset to get files for '--input_path' parameter
// Nextflow is unable to recursively download directories via HTTPS
if (workflow.profile.contains('test')) {
if (!isOffline()) {
process GetTestData {
output:
file "test-datasets/fast5/$barcoded/" into ch_input_path
script:
barcoded = workflow.profile.contains('test_nonbc') ? "nonbarcoded" : "barcoded"
"""
git clone https://github.com/nf-core/test-datasets.git --branch nanoseq --single-branch
"""
}
} else {
exit 1, "NXF_OFFLINE=true or -offline has been set so cannot download and run any test dataset!"
}
} else {
if (params.input_path) { ch_input_path = Channel.fromPath(params.input_path, checkIfExists: true) } else { exit 1, "Please specify a valid run directory to perform basecalling!" }
}
// Need to stage guppy_config properly depending on whether its a file or string
if (!params.guppy_config) {
if (!params.flowcell) { exit 1, "Please specify a valid flowcell identifier for basecalling!" }
if (!params.kit) { exit 1, "Please specify a valid kit identifier for basecalling!" }
} else if (file(params.guppy_config).exists()) {
ch_guppy_config = Channel.fromPath(params.guppy_config)
}
// Need to stage guppy_model properly depending on whether its a file or string
if (params.guppy_model) {
if (file(params.guppy_model).exists()) {
ch_guppy_model = Channel.fromPath(params.guppy_model)
}
}
} else {
if (!params.skip_demultiplexing) {
if (!params.barcode_kit) {
params.barcode_kit = 'Auto'
}
def qcatBarcodeKitList = ['Auto', 'RBK001', 'RBK004', 'NBD103/NBD104',
'NBD114', 'NBD104/NBD114', 'PBC001', 'PBC096',
'RPB004/RLB001', 'PBK004/LWB001', 'RAB204', 'VMK001', 'DUAL']
if (params.barcode_kit && qcatBarcodeKitList.contains(params.barcode_kit)) {
if (params.input_path) { ch_input_path = Channel.fromPath(params.input_path, checkIfExists: true) } else { exit 1, "Please specify a valid input fastq file to perform demultiplexing!" }
} else {
exit 1, "Please provide a barcode kit to demultiplex with qcat. Valid options: ${qcatBarcodeKitList}"
}
}
}
if (!params.skip_alignment) {
if (params.aligner != 'minimap2' && params.aligner != 'graphmap2') {
exit 1, "Invalid aligner option: ${params.aligner}. Valid options: 'minimap2', 'graphmap2'"
}
if (params.protocol != 'DNA' && params.protocol != 'cDNA' && params.protocol != 'directRNA') {
exit 1, "Invalid protocol option: ${params.protocol}. Valid options: 'DNA', 'cDNA', 'directRNA'"
}
}
if (!params.skip_transcriptquant) {
if (params.transcriptquant != 'bambu' && params.transcriptquant != 'stringtie2') {
exit 1, "Invalid transcript quantification option: ${params.transcriptquant}. Valid options: 'bambu', 'stringtie2'"
}
if (params.protocol != 'cDNA' && params.protocol != 'directRNA') {
exit 1, "Invalid protocol option: ${params.protocol}. Valid options: 'cDNA', 'directRNA'"
}
}
// Stage config files
ch_multiqc_config = file("$baseDir/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("$baseDir/docs/output.md", checkIfExists: true)
ch_output_docs_images = file("$baseDir/docs/images/", checkIfExists: true)
// 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 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
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
summary['Stranded'] = (params.stranded || params.protocol == 'directRNA') ? 'Yes' : 'No'
summary['Skip Basecalling'] = params.skip_basecalling ? 'Yes' : 'No'
summary['Skip Demultiplexing'] = params.skip_demultiplexing ? 'Yes' : 'No'
if (!params.skip_basecalling) {
summary['Run Dir'] = params.input_path
summary['Flowcell ID'] = params.flowcell ?: 'Not required'
summary['Kit ID'] = params.kit ?: 'Not required'
summary['Barcode Kit ID'] = params.barcode_kit ?: 'Unspecified'
summary['Guppy Config File'] = params.guppy_config ?: 'Unspecified'
summary['Guppy Model File'] = params.guppy_model ?:'Unspecified'
summary['Guppy GPU Mode'] = params.guppy_gpu ? 'Yes' : 'No'
summary['Guppy GPU Runners'] = params.guppy_gpu_runners
summary['Guppy CPU Threads'] = params.guppy_cpu_threads
summary['Guppy GPU Device'] = params.gpu_device ?: 'Unspecified'
summary['Guppy GPU Options'] = params.gpu_cluster_options ?: 'Unspecified'
}
if (params.skip_basecalling && !params.skip_demultiplexing) {
summary['Input FastQ File'] = params.input_path
summary['Barcode Kit ID'] = params.barcode_kit ?: 'Unspecified'
summary['Qcat Min Score'] = params.qcat_min_score
summary['Qcat Detect Middle'] = params.qcat_detect_middle ? 'Yes': 'No'
}
if (!params.skip_alignment) {
summary['Aligner'] = params.aligner
summary['Save Intermeds'] = params.save_align_intermeds ? 'Yes' : 'No'
}
if (!params.skip_transcriptquant && params.protocol != 'DNA') {
summary['Transcript Quantification'] = params.transcriptquant
}
if (params.skip_alignment) summary['Skip Alignment'] = 'Yes'
if (params.skip_bigbed) summary['Skip BigBed'] = 'Yes'
if (params.skip_bigwig) summary['Skip BigWig'] = 'Yes'
if (params.skip_qc) summary['Skip QC'] = 'Yes'
if (params.skip_pycoqc) summary['Skip PycoQC'] = 'Yes'
if (params.skip_nanoplot) summary['Skip NanoPlot'] = '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 - $workflow.container"
summary['Output dir'] = params.outdir
summary['Launch dir'] = workflow.launchDir
summary['Working dir'] = workflow.workDir
summary['Script dir'] = workflow.projectDir
summary['User'] = workflow.userName
if (workflow.profile.contains('awsbatch')) {
summary['AWS Region'] = params.awsregion
summary['AWS Queue'] = params.awsqueue
summary['AWS CLI'] = params.awscli
}
summary['Config Profile'] = workflow.profile
if (params.config_profile_description) summary['Config Description'] = params.config_profile_description
if (params.config_profile_contact) summary['Config Contact'] = params.config_profile_contact
if (params.config_profile_url) summary['Config URL'] = params.config_profile_url
if (params.email || params.email_on_fail) {
summary['E-mail Address'] = params.email
summary['E-mail on failure'] = params.email_on_fail
summary['MultiQC maxsize'] = params.max_multiqc_email_size
}
log.info summary.collect { k,v -> "${k.padRight(19)}: $v" }.join("\n")
log.info "-\033[2m--------------------------------------------------\033[0m-"
// Check the hostnames against configured profiles
checkHostname()
/*
* PREPROCESSING - CHECK SAMPLESHEET
*/
process CheckSampleSheet {
tag "$samplesheet"
publishDir "${params.outdir}/pipeline_info", mode: 'copy'
input:
file samplesheet from ch_input
output:
file "*reformat.csv" into ch_samplesheet_reformat
file "total_conditions.csv" into ch_sample_condition
script: // This script is bundled with the pipeline, in nf-core/nanoseq/bin/
"""
check_samplesheet.py $samplesheet samplesheet_reformat.csv
"""
}
// Function to resolve fasta and gtf file if using iGenomes
// Returns [ sample, fastq, barcode, fasta, gtf, is_transcripts, annotation_str ]
def get_sample_info(LinkedHashMap sample, LinkedHashMap genomeMap) {
// Resolve fasta and gtf file if using iGenomes
def fasta = false
def gtf = false
if (sample.genome) {
if (genomeMap.containsKey(sample.genome)) {
fasta = file(genomeMap[sample.genome].fasta, checkIfExists: true)
gtf = file(genomeMap[sample.genome].gtf, checkIfExists: true)
} else {
fasta = file(sample.genome, checkIfExists: true)
}
}
// Check if fastq and gtf file exists
sample_path = sample.sample_path ? file(sample.sample_path, checkIfExists: true) : null
gtf = sample.gtf ? file(sample.gtf, checkIfExists: true) : gtf
return [ sample.sample, sample_path, sample.barcode, fasta, gtf, sample.is_transcripts.toBoolean(), fasta.toString()+';'+gtf.toString() ]
}
// Create channels = [ sample, barcode, fasta, gtf, is_transcripts, annotation_str ]
ch_samplesheet_reformat
.splitCsv(header:true, sep:',')
.map { get_sample_info(it, params.genomes) }
.map { it -> [ it[0], it[2], it[3], it[4], it[5], it[6] ] }
.into { ch_sample_info;
ch_sample_name;
ch_transquant_info}
ch_sample_condition
.splitCsv(header:false, sep:',')
.map {it -> it.size()}
.into { ch_deseq2_num_condition;
ch_dexseq_num_condition}
if (!params.skip_basecalling) {
// Get sample name for single sample when --skip_demultiplexing
ch_sample_name
.first()
.map { it[0] }
.set { ch_sample_name }
/*
* STEP 1 - Basecalling and demultipexing using Guppy
*/
process Guppy {
tag "$input_path"
label 'process_high'
publishDir path: "${params.outdir}/guppy", mode: 'copy',
saveAs: { filename ->
if (!filename.endsWith("guppy.txt")) filename
}
input:
file input_path from ch_input_path
val name from ch_sample_name
file guppy_config from ch_guppy_config.ifEmpty([])
file guppy_model from ch_guppy_model.ifEmpty([])
output:
file "fastq/*.fastq.gz" into ch_fastq
file "basecalling/*.txt" into ch_guppy_pycoqc_summary,
ch_guppy_nanoplot_summary
file "basecalling/*"
file "v_guppy.txt" into ch_guppy_version
script:
barcode_kit = params.barcode_kit ? "--barcode_kits $params.barcode_kit" : ""
proc_options = params.guppy_gpu ? "--device $params.gpu_device --num_callers $task.cpus --cpu_threads_per_caller $params.guppy_cpu_threads --gpu_runners_per_device $params.guppy_gpu_runners" : "--num_callers 2 --cpu_threads_per_caller ${task.cpus/2}"
def config = "--flowcell $params.flowcell --kit $params.kit"
if (params.guppy_config) config = file(params.guppy_config).exists() ? "--config ./$guppy_config" : "--config $params.guppy_config"
def model = ""
if (params.guppy_model) model = file(params.guppy_model).exists() ? "--model ./$guppy_model" : "--model $params.guppy_model"
"""
guppy_basecaller \\
--input_path $input_path \\
--save_path ./basecalling \\
--records_per_fastq 0 \\
--compress_fastq \\
$barcode_kit \\
$proc_options \\
$config \\
$model
guppy_basecaller --version &> v_guppy.txt
## Concatenate fastq files
mkdir fastq
cd basecalling
if [ "\$(find . -type d -name "barcode*" )" != "" ]
then
for dir in barcode*/
do
dir=\${dir%*/}
cat \$dir/*.fastq.gz > ../fastq/\$dir.fastq.gz
done
else
cat *.fastq.gz > ../fastq/${name}.fastq.gz
fi
"""
}
// Create channels = [ sample, fastq, fasta, gtf, is_transcripts, annotation_str ]
ch_fastq
.flatten()
.map { it -> [ it, it.baseName.substring(0,it.baseName.lastIndexOf('.')) ] } // [barcode001.fastq, barcode001]
.join(ch_sample_info, by: 1) // join on barcode
.map { it -> [ it[2], it[1], it[3], it[4], it[5], it[6] ] }
.into { ch_fastq_nanoplot;
ch_fastq_fastqc;
ch_fastq_sizes;
ch_fastq_gtf;
ch_fastq_index;
ch_fastq_align }
} else {
if (!params.skip_demultiplexing) {
/*
* STEP 1 - Demultipexing using qcat
*/
process Qcat {
tag "$input_path"
label 'process_medium'
publishDir path: "${params.outdir}/qcat", mode: 'copy'
input:
file input_path from ch_input_path
output:
file "fastq/*.fastq.gz" into ch_fastq
script:
detect_middle = params.qcat_detect_middle ? "--detect-middle $params.qcat_detect_middle" : ""
"""
## Unzip fastq file
## qcat doesnt support zipped files yet
FILE=$input_path
if [[ \$FILE == *.gz ]]
then
zcat $input_path > unzipped.fastq
FILE=unzipped.fastq
fi
qcat \\
-f \$FILE \\
-b ./fastq \\
--kit $params.barcode_kit \\
--min-score $params.qcat_min_score \\
$detect_middle
## Zip fastq files
pigz -p $task.cpus fastq/*
"""
}
// Create channels = [ sample, fastq, fasta, gtf, is_transcripts, annotation_str ]
ch_fastq
.flatten()
.map { it -> [ it, it.baseName.substring(0,it.baseName.lastIndexOf('.'))] } // [barcode001.fastq, barcode001]
.join(ch_sample_info, by: 1) // join on barcode
.map { it -> [ it[2], it[1], it[3], it[4], it[5], it[6] ] }
.into { ch_fastq_nanoplot;
ch_fastq_fastqc;
ch_fastq_sizes;
ch_fastq_gtf;
ch_fastq_index;
ch_fastq_align }
} else {
if (!params.skip_alignment) {
// Create channels = [ sample, fastq, fasta, gtf, is_transcripts, annotation_str ]
ch_samplesheet_reformat
.splitCsv(header:true, sep:',')
.map { get_sample_info(it, params.genomes) }
.map { it -> [ it[0], it[1], it[3], it[4], it[5], it[6] ] }
.into { ch_fastq_nanoplot;
ch_fastq_fastqc;
ch_fastq_sizes;
ch_fastq_gtf;
ch_fastq_index;
ch_fastq_align }
} else {
ch_fastq_nanoplot = Channel.empty()
ch_fastq_fastqc = Channel.empty()
}
}
ch_guppy_version = Channel.empty()
ch_guppy_pycoqc_summary = Channel.empty()
ch_guppy_nanoplot_summary = Channel.empty()
}
/*
* STEP 2 - QC using PycoQC
*/
process PycoQC {
tag "$summary_txt"
label 'process_low'
publishDir "${params.outdir}/pycoqc", mode: 'copy'
when:
!params.skip_basecalling && !params.skip_qc && !params.skip_pycoqc
input:
file summary_txt from ch_guppy_pycoqc_summary
output:
file "*.html"
script:
"""
pycoQC -f $summary_txt -o pycoQC_output.html
"""
}
/*
* STEP 3 - QC using NanoPlot
*/
process NanoPlotSummary {
tag "$summary_txt"
label 'process_low'
publishDir "${params.outdir}/nanoplot/summary", mode: 'copy'
when:
!params.skip_basecalling && !params.skip_qc && !params.skip_nanoplot
input:
file summary_txt from ch_guppy_nanoplot_summary
output:
file "*.{png,html,txt,log}"
script:
"""
NanoPlot -t $task.cpus --summary $summary_txt
"""
}
/*
* STEP 4 - FastQ QC using NanoPlot
*/
process NanoPlotFastQ {
tag "$sample"
label 'process_low'
publishDir "${params.outdir}/nanoplot/fastq/${sample}", mode: 'copy'
when:
!params.skip_qc && !params.skip_nanoplot
input:
set val(sample), file(fastq) from ch_fastq_nanoplot.map { ch -> [ ch[0], ch[1] ] }
output:
file "*.{png,html,txt,log}"
script:
"""
NanoPlot -t $task.cpus --fastq $fastq
"""
}
/*
* STEP 5 - FastQ QC using FastQC
*/
process FastQC {
tag "$sample"
label 'process_medium'
publishDir "${params.outdir}/fastqc", mode: 'copy'
when:
!params.skip_qc && !params.skip_fastqc
input:
set val(sample), file(fastq) from ch_fastq_fastqc.map { ch -> [ ch[0], ch[1] ] }
output:
file "*.{zip,html}" into ch_fastqc_mqc
script:
"""
[ ! -f ${sample}.fastq.gz ] && ln -s $fastq ${sample}.fastq.gz
fastqc -q -t $task.cpus ${sample}.fastq.gz
"""
}
if (!params.skip_alignment) {
// Get unique list of all fasta files
ch_fastq_sizes
.filter { it[2] }
.map { it -> [ it[2], it[-1].toString() ] } // [ fasta, annotation_str ]
.unique()
.set { ch_fastq_sizes }
/*
* STEP 6 - Make chromosome sizes file
*/
process GetChromSizes {
tag "$fasta"
input:
set file(fasta), val(name) from ch_fastq_sizes
output:
set file("*.sizes"), val(name) into ch_chrom_sizes
script:
"""
samtools faidx $fasta
cut -f 1,2 ${fasta}.fai > ${fasta}.sizes
"""
}
// Get unique list of all gtf files
ch_fastq_gtf
.filter { it[3] }
.map { it -> [ it[3], it[-1] ] } // [ gtf, annotation_str ]
.unique()
.set { ch_fastq_gtf }
/*
* STEP 7 - Convert GTF to BED12
*/
process GTFToBED {
tag "$gtf"
label 'process_low'
input:
set file(gtf), val(name) from ch_fastq_gtf
output:
set file("*.bed"), val(name) into ch_gtf_bed
script: // This script is bundled with the pipeline, in nf-core/nanoseq/bin/
"""
gtf2bed $gtf > ${gtf.baseName}.bed
"""
}
ch_chrom_sizes
.join(ch_gtf_bed, by: 1, remainder:true)
.map { it -> [ it[1], it[2], it[0] ] }
.cross(ch_fastq_index) { it -> it[-1] }
.flatten()
.collate(9)
.map { it -> [ it[5], it[0], it[6], it[1], it[7], it[8] ]} // [ fasta, sizes, gtf, bed, is_transcripts, annotation_str ]
.unique()
.set { ch_fasta_index }
/*
* STEP 8 - Create genome/transcriptome index
*/
if (params.aligner == 'minimap2') {
process MiniMap2Index {
tag "$fasta"
label 'process_medium'
input:
set file(fasta), file(sizes), val(gtf), val(bed), val(is_transcripts), val(annotation_str) from ch_fasta_index
output:
set file(fasta), file(sizes), val(gtf), val(bed), val(is_transcripts), file("*.mmi"), val(annotation_str) into ch_index
script:
preset = (params.protocol == 'DNA' || is_transcripts) ? "-ax map-ont" : "-ax splice"
kmer = (params.protocol == 'directRNA') ? "-k14" : ""
stranded = (params.stranded || params.protocol == 'directRNA') ? "-uf" : ""
// TODO pipeline: Should be staging bed file properly as an input
junctions = (params.protocol != 'DNA' && bed) ? "--junc-bed ${file(bed)}" : ""
"""
minimap2 $preset $kmer $stranded $junctions -t $task.cpus -d ${fasta}.mmi $fasta
"""
}
} else {
process GraphMap2Index {
tag "$fasta"
label 'process_high'
input:
set file(fasta), file(sizes), val(gtf), val(bed), val(is_transcripts), val(annotation_str) from ch_fasta_index
output:
set file(fasta), file(sizes), val(gtf), val(bed), val(is_transcripts), file("*.gmidx"), val(annotation_str) into ch_index
script:
preset = (params.protocol == 'DNA' || is_transcripts) ? "" : "-x rnaseq"
// TODO pipeline: Should be staging gtf file properly as an input
junctions = (params.protocol != 'DNA' && !is_transcripts && gtf) ? "--gtf ${file(gtf)}" : ""
"""
graphmap2 align $preset $junctions -t $task.cpus -I -r $fasta
"""
}
}
ch_index
.cross(ch_fastq_align) { it -> it[-1] }
.flatten()
.collate(13)
.map { it -> [ it[7], it[8], it[0], it[1], it[2], it[3], it[4], it[5] ]} // [ sample, fastq, fasta, sizes, gtf, bed, is_transcripts, index ]
.set { ch_index }
/*
* STEP 9 - Align fastq files
*/
if (params.aligner == 'minimap2') {
process MiniMap2Align {
tag "$sample"
label 'process_medium'
if (params.save_align_intermeds) {
publishDir path: "${params.outdir}/${params.aligner}", mode: 'copy',
saveAs: { filename ->
if (filename.endsWith(".sam")) filename
}
}
input:
set val(sample), file(fastq), file(fasta), file(sizes), val(gtf), val(bed), val(is_transcripts), file(index) from ch_index
output:
set val(sample), file(sizes), val(is_transcripts), file("*.sam") into ch_align_sam
script:
preset = (params.protocol == 'DNA' || is_transcripts) ? "-ax map-ont" : "-ax splice"
kmer = (params.protocol == 'directRNA') ? "-k14" : ""
stranded = (params.stranded || params.protocol == 'directRNA') ? "-uf" : ""
// TODO pipeline: Should be staging bed file properly as an input
junctions = (params.protocol != 'DNA' && bed) ? "--junc-bed ${file(bed)}" : ""
"""
minimap2 $preset $kmer $stranded $junctions -t $task.cpus $index $fastq > ${sample}.sam
"""
}
} else {
process GraphMap2Align {
tag "$sample"
label 'process_medium'
if (params.save_align_intermeds) {
publishDir path: "${params.outdir}/${params.aligner}", mode: 'copy',
saveAs: { filename ->
if (filename.endsWith(".sam")) filename
}
}
input:
set val(sample), file(fastq), file(fasta), file(sizes), val(gtf), val(bed), val(is_transcripts), file(index) from ch_index
output:
set val(sample), file(sizes), val(is_transcripts), file("*.sam") into ch_align_sam
script:
preset = (params.protocol == 'DNA' || is_transcripts) ? "" : "-x rnaseq"
// TODO pipeline: Should be staging gtf file properly as an input
junctions = (params.protocol != 'DNA' && !is_transcripts && gtf) ? "--gtf ${file(gtf)}" : ""
"""
graphmap2 align $preset $junctions -t $task.cpus -r $fasta -i $index -d $fastq -o ${sample}.sam --extcigar
"""
}
}
/*
* STEP 10 - Coordinate sort BAM files
*/
process SortBAM {
tag "$sample"
label 'process_medium'
publishDir path: "${params.outdir}/${params.aligner}", mode: 'copy',
saveAs: { filename ->
if (filename.endsWith(".flagstat")) "samtools_stats/$filename"
else if (filename.endsWith(".idxstats")) "samtools_stats/$filename"
else if (filename.endsWith(".stats")) "samtools_stats/$filename"
else if (filename.endsWith(".sorted.bam")) "bam/$filename"
else if (filename.endsWith(".sorted.bam.bai")) "bam_index/$filename"
else null
}
input:
set val(sample), file(sizes), val(is_transcripts), file(sam) from ch_align_sam
output:
set val(sample), file(sizes), val(is_transcripts), file("*.sorted.{bam,bam.bai}") into ch_sortbam_bedgraph,
ch_sortbam_bed12
set val(sample), file("*.sorted.bam") into ch_sortbam_stringtie
file "*.sorted.bam" into ch_bamlist
file "*.{flagstat,idxstats,stats}" into ch_sortbam_stats_mqc
script:
"""
samtools view -b -h -O BAM -@ $task.cpus -o ${sample}.bam $sam
samtools sort -@ $task.cpus -o ${sample}.sorted.bam -T $sample ${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 > ${sample}.sorted.bam.stats
"""
}
} else {
ch_sortbam_bedgraph = Channel.empty()
ch_sortbam_bed12 = Channel.empty()
ch_sortbam_stats_mqc = Channel.empty()
ch_samplesheet_reformat
.splitCsv(header:true, sep:',')
.map { get_sample_info(it, params.genomes) }
.map { it -> [ it[0], it[1]] }
.into {ch_sortbam_stringtie;
ch_get_bams}
ch_get_bams
.map {it -> it[1]}
.set{ch_bamlist}
}
/*
* STEP 11 - Convert BAM to BEDGraph
*/
process BAMToBedGraph {
tag "$sample"
label 'process_medium'
when:
!params.skip_alignment && !params.skip_bigwig
input:
set val(sample), file(sizes), val(is_transcripts), file(bam) from ch_sortbam_bedgraph
output:
set val(sample), file(sizes), val(is_transcripts), file("*.bedGraph") into ch_bedgraph
script:
split = (params.protocol == 'DNA' || is_transcripts) ? "" : "-split"
"""
bedtools genomecov $split -ibam ${bam[0]} -bg | sort -k1,1 -k2,2n > ${sample}.bedGraph
"""
}
/*
* STEP 12 - Convert BEDGraph to BigWig
*/
process BedGraphToBigWig {
tag "$sample"
label 'process_medium'
publishDir path: "${params.outdir}/${params.aligner}/bigwig/", mode: 'copy'
when:
!params.skip_alignment && !params.skip_bigwig
input:
set val(sample), file(sizes), val(is_transcripts), file(bedgraph) from ch_bedgraph
output:
set val(sample), file(sizes), val(is_transcripts), file("*.bigWig") into ch_bigwig
script:
"""
bedGraphToBigWig $bedgraph $sizes ${sample}.bigWig
"""
}
/*
* STEP 13 - Convert BAM to BED12
*/
process BAMToBed12 {
tag "$sample"
label 'process_medium'
when:
!params.skip_alignment && !params.skip_bigbed && (params.protocol == 'directRNA' || params.protocol == 'cDNA')
input:
set val(sample), file(sizes), val(is_transcripts), file(bam) from ch_sortbam_bed12
output:
set val(sample), file(sizes), val(is_transcripts), file("*.bed12") into ch_bed12
script:
"""
bedtools bamtobed -bed12 -cigar -i ${bam[0]} | sort -k1,1 -k2,2n > ${sample}.bed12
"""
}
/*
* STEP 14 - Convert BED12 to BigBED
*/
process Bed12ToBigBed {
tag "$sample"
label 'process_medium'
publishDir path: "${params.outdir}/${params.aligner}/bigbed/", mode: 'copy'
when:
!params.skip_alignment && !params.skip_bigbed && (params.protocol == 'directRNA' || params.protocol == 'cDNA')
input:
set val(sample), file(sizes), val(is_transcripts), file(bed12) from ch_bed12
output:
set val(sample), file(sizes), val(is_transcripts), file("*.bigBed") into ch_bigbed
script:
"""
bedToBigBed $bed12 $sizes ${sample}.bigBed
"""
}
if (!params.skip_transcriptquant) {
/*
* STEP 15 - Transcript Quantification
*/
if (params.transcriptquant == 'bambu') {
ch_transquant_info
.map {it -> [it[2],it[3]]}
.set { ch_bambu_input }
params.Bambuscript= "$baseDir/bin/runBambu.R"
ch_Bambuscript = Channel.fromPath("$params.Bambuscript", checkIfExists:true)
process Bambu {
tag "$sample"
label 'process_medium'
publishDir "${params.outdir}/${params.transcriptquant}", mode: 'copy',
saveAs: { filename ->
if (!filename.endsWith(".version")) filename
}
input:
set file(genomeseq), file(annot) from ch_bambu_input
file bams from ch_bamlist.collect()
file Bambuscript from ch_Bambuscript
output:
file "counts_gene.txt" into ch_deseq2_in
file "counts_transcript.txt" into ch_dexseq_in
file "extended_annotations.gtf"
script:
"""
Rscript --vanilla $Bambuscript --tag=. --ncore=$task.cpus --annotation=$annot --fasta=$genomeseq $bams
"""
}
} else {
ch_transquant_info
.map {it -> [it[0],it[2], it[3]]}
.set{ch_fasta_gtf}
ch_fasta_gtf
.join(ch_sortbam_stringtie)
.set{ch_txome_reconstruction}
process StringTie2 {
tag "$sample"
label 'process_medium'
publishDir "${params.outdir}/${params.transcriptquant}", mode: 'copy',
saveAs: { filename ->
if (!filename.endsWith(".version")) filename
}
input:
set val(name), val(genomeseq), file(annot), file(bam) from ch_txome_reconstruction
output:
set val(name), file(bam) into ch_txome_feature_count
file annot into ch_annot
file("*.version") into ch_stringtie_version
val "${params.outdir}/${params.transcriptquant}" into ch_stringtie_outputs
file "*.out.gtf" into ch_gtflist
script:
"""
stringtie -L -G $annot -o ${name}.out.gtf $bam
stringtie --version &> stringtie.version
"""
}
ch_stringtie_outputs
.unique()
.set {ch_stringtie_dir}
ch_annot
.unique()
.set{ch_annotation}
process StringTie2Merge {
tag "$sample"
label 'process_medium'
publishDir "${params.outdir}/${params.transcriptquant}", mode: 'copy',
saveAs: { filename ->
if (!filename.endsWith(".version")) filename
}
input:
val stringtie_dir from ch_stringtie_dir
file gtfs from ch_gtflist.collect()
file annot from ch_annotation
output:
file "merged.combined.gtf" into ch_merged_gtf
script:
"""
stringtie --merge $gtfs -G $annot -o merged.combined.gtf
"""
}
process FeatureCounts {
tag "$sample"
label 'process_medium'
publishDir "${params.outdir}/${params.transcriptquant}", mode: 'copy',
saveAs: { filename ->
if (!filename.endsWith(".version")) "featureCounts/$filename"
}
input:
file annot from ch_merged_gtf
file bams from ch_bamlist.collect()
output:
file("*.version") into ch_feat_counts_version