nextflow - spltiCSV - each element - error : If you need to reuse the same component

For a nextflow pipeline I'd like to read in a CSV file with five columns:

sample1,path/normal_R1.fastq,path/normal_R2.fastq,path/tumor_R1.fastq,path/tumor_R2.fastq
sample2,path/normal_R1.fastq,path/normal_R2.fastq,path/tumor_R1.fastq,path/tumor_R2.fastq

I read in the file, create a linkedHashMap. For each element I'd like to run few processes. The processes has been working fine without CSV iteration, as they were provided by a channel of tumor files and a channel of normal files.

When I edit the code with a CSV, I get error as:

> Process 'FASTP' has been already used -- If you need to reuse the same
> component, include it with a different name or include it in a
> different workflow context

Below is the code:

include { FASTP} from './fastp_process.nf'
include {bwa_index} from './index_process.nf'
include { align_bwa_mem} from './bwamem_process_already_index.nf'
include { gatk_markduplicates} from './gatk_markduplicates_process.nf'
include {setupnmdtags} from './setupnmdtags_process.nf'
include { recalibrate_bam } from './recalibratebam_process.nf'
include { applybqsr } from './applybqsr_process.nf'
include { mutect2 } from './mutect2_process.nf'
include { lancet } from './lancet_process.nf'
include { manta } from './manta_process.nf'
include { strelka } from './strelka_process.nf'
include { gatk_merge_vcfs } from  './gatk_merge_vcfs.nf'
workflow {
def csvFile = file("input_nextflow_files.csv")
def csvLines = csvFile.text.readLines()
def sampleMap = csvLines.collectEntries { line ->
def lineCols = line.split(',')
if (lineCols.size() >= 5) {
def sampleName = lineCols[0]
def normalR1 = file(lineCols[1])
def normalR2 = file(lineCols[2])
def tumorR1 = file(lineCols[3])
def tumorR2 = file(lineCols[4])
[(sampleName): [tuple(normalR1, normalR2), tuple(tumorR1, tumorR2)]]
} else {
return [:]
}
}
sampleMap.each { sampleName, pairList ->
def normalPair = pairList[0]
def tumorPair = pairList[1]
FASTP(tumorPair,normalPair,sampleName)
align_bwa_mem(FASTP.out.reads_tumor,FASTP.out.reads_normal) //already_created index
}
}

I believe it is something related to the FASTP process below (input):

process FASTP {
maxForks 3
debug true
input:
path(reads_tumor)  //val outdir  //doesn't work with path (outdir) // we pass multiple reads - for tumor and normal
path(reads_normal)  //val outdir  //doesn't work with path (outdir)
val (sample_name)
output:
tuple val(sample_name), path("${sample_id_tumor}_trim_{1,2}.fq.gz"), emit: reads_tumor
path("${sample_id_tumor}.fastp.json"), emit: json_tumor
path("${sample_id_tumor}.fastp.html"), emit: html_tumor
tuple val(sample_id_normal), path("${sample_id_normal}_trim_{1,2}.fq.gz"), emit: reads_normal
path("${sample_id_normal}.fastp.json"), emit: json_normal
path("${sample_id_normal}.fastp.html"), emit: html_normal
script:
def (r1_normal, r2_normal) = reads_normal
def (r1_tumor, r2_tumor)=reads_tumor
"""
ml fastp
fastp  --in1 "${r1_normal}" --in2 "${r2_normal}" -q 20  -u 20 -l 40 --detect_adapter_for_pe --out1 "${sample_id_normal}_trim_1.fq.gz" --out2 "${sample_id_normal}_trim_2.fq.gz" --json "${sample_id_normal}.fastp.json" --html "${sample_id_normal}.fastp.html" --thread 12 
fastp  --in1 "${r1_tumor}" --in2 "${r2_tumor}" -q 20  -u 20 -l 40 --detect_adapter_for_pe --out1 "${sample_id_tumor}_trim_1.fq.gz" --out2 "${sample_id_tumor}_trim_2.fq.gz" --json "${sample_id_tumor}.fastp.json" --html "${sample_id_tumor}.fastp.html" --thread 12 
echo "Exiting fastp"
"""
}

I do not know how to fix this error. I checked if multiple times I'm not including FASTP process it is fine. I remove include and FASTP calling process they didn't work. So I cannot figure out what's going on.

When you iterate through your sample map using each, you are effectively trying to re-use the FASTP and align_bwa_mem processes with each iteration. Nextflow just complains that if they (i.e. the processes) need to be re-used, they'll need to be included using a different name (i.e. using a module alias) or in a different workflow context (i.e. using a sub-workflow). A better way to achieve what you want is to use channels and the splitCSV operator, for example:

params.samples_csv = 'input_nextflow_files.csv'
include { FASTP } from './fastp_process.nf'
workflow {
def header = ['sampleName', 'normalR1', 'normalR2', 'tumorR1', 'tumorR2']
Channel
.fromPath( params.samples_csv )
.splitCsv( header: header )
.multiMap { row ->
def tumor_reads = tuple( file(row.tumorR1), file(row.tumorR2) )
def normal_reads = tuple( file(row.normalR1), file(row.normalR2) )
tumor:
tuple( row.sampleName, tumor_reads )
normal:
tuple( row.sampleName, normal_reads )
}
.set { samples }
FASTP( samples.tumor.mix( samples.normal ) )
...
}

Or if you wanted more flexibility, another way would be to import FASTP using a module alias:

params.samples_csv = 'input_nextflow_files.csv'
include { FASTP as FASTP_TUMOR } from './fastp_process.nf'
include { FASTP as FASTP_NORMAL } from './fastp_process.nf'
workflow {
...
FASTP_TUMOR( samples.tumor )
FASTP_NORMAL( samples.normal )
...
}

Contents of ./fastp_process.nf:

process FASTP {
tag { sample_id }
input:
tuple val(sample_id), path(reads, stageAs: 'reads/*')
output:
tuple val(sample_id), path("${sample_id}_trim_{1,2}.fq.gz"), emit: reads
path "${sample_id}.fastp.json", emit: json
path "${sample_id}.fastp.html", emit: html
script:
def (r1, r2) = reads
"""
fastp \\
--in1 "${r1}" \\
--in2 "${r2}" \\
-q 20 \\
-u 20 \\
-l 40 \\
--detect_adapter_for_pe \\
--out1 "${sample_id}_trim_1.fq.gz" \\
--out2 "${sample_id}_trim_2.fq.gz" \\
--json "${sample_id}.fastp.json" \\
--html "${sample_id}.fastp.html" \\
--thread {task.cpus}
"""
}