diff --git a/workflow/main.nf b/workflow/main.nf
index 373469854d65643314e08163b2ed69972d607897..41622759eee0e4c74157d9ca4b403efa91ae3833 100644
--- a/workflow/main.nf
+++ b/workflow/main.nf
@@ -3,39 +3,6 @@
// Default parameter values to run tests
params.input = "$baseDir"
params.output= "$baseDir/output"
-
-params.files="$params.input/*.f*"
-files=Channel.fromPath(params.files)
-
-process checkinputfiles {
-
- input:
-
- file fqs from files
-
- output:
-
- file("*.fastq.gz") into fastqs
-
- script:
-
- """
- if [[ $fqs == *.fq ]];
- then new_name=`echo ${fqs} | sed -e "s/.fq\$/.fastq/"`;
- mv ${fqs} \${new_name};
- gzip -f \${new_name};
- elif [[ $fqs == *.fastq ]];
- then gzip -f $fqs;
- elif [[ $fqs == *.fq.gz ]];
- then new_name=`echo ${fqs} | sed -e "s/.fq.gz\$/.fastq.gz/"`;
- mv ${fqs} \${new_name};
- else
- zcat ${fqs} | gzip -c > ${fqs}.temp
- mv ${fqs}.temp ${fqs}
- fi;
- """
-}
-
params.design="$params.input/design.txt"
params.genome="/project/shared/bicf_workflow_ref/human/grch38/"
params.markdups="picard"
@@ -46,7 +13,6 @@ params.align = 'hisat'
params.fusion = 'skip'
params.dea = 'detect'
-design_file = file(params.design)
design_file = file(params.design)
gtf_file = file("$params.genome/gencode.gtf")
genenames = file("$params.genome/genenames.txt")
@@ -57,19 +23,61 @@ indel="$params.genome/goldindels.vcf.gz"
knownindel=file(indel)
dbsnp=file(dbsnp)
+files = Channel
+ .fromPath("$params.input/*")
+good = Channel.fromPath("$params.input/*.fastq.gz")
+
+process checkinputfiles {
+ module 'parallel/20150122:pigz/2.4'
+ queue 'super'
+
+ input:
+
+ file ("*") from files.collect()
+ file design_file name "design.tsv"
+
+ output:
+
+ set file ("design.tsv"), file ("*.fastq.gz") into design
+ file ("*.fastq.gz") into fastqs
+
+ script:
+
+ """
+ for fqs in `ls | grep -v "^design.tsv\$"`;
+ do if [[ \$fqs == *.fq ]];
+ then new_name=`echo \${fqs} | sed -e "s/.fq\$/.fastq/"`;
+ mv \${fqs} \${new_name};
+ echo "pigz -f \${new_name}";
+ elif [[ \$fqs == *.fastq ]];
+ then echo "pigz -f \$fqs";
+ elif [[ \$fqs == *.fq.gz ]];
+ then new_name=`echo \${fqs} | sed -e "s/.fq.gz\$/.fastq.gz/"`;
+ mv \${fqs} \${new_name};
+ fi;
+ done | shuf | parallel -j\${SLURM_CPUS_ON_NODE};
+ """
+}
+
// params genome is the directory
// base name for the index is always genome
index_path = file(params.genome)
process checkdesignfile {
+ executor 'local'
publishDir "$params.output", mode: 'copy'
+
input:
- file design_file name 'design.ori.txt'
+
+ set file ("design.ori.txt"), file ("*") from design
output:
+
file("design.valid.txt") into newdesign
stdout spltnames
+
script:
+
"""
perl -p -e 's/\\r\\n*/\\n/g' design.ori.txt > design.fix.txt
perl $baseDir/scripts/check_designfile.pl ${params.pairs} design.fix.txt
@@ -78,14 +86,17 @@ process checkdesignfile {
def fileMap = [:]
-fastqs.each {
+fastqs
+ .mix(good)
+ .flatten()
+ .each {
final fileName = it.getFileName().toString()
prefix = fileName.lastIndexOf('/')
fileMap[fileName] = it
}
if (params.pairs == 'pe') {
-spltnames
+ spltnames
.splitCsv()
.filter { fileMap.get(it[1]) != null & fileMap.get(it[2]) != null }
.map { it -> tuple(it[0], fileMap.get(it[1]), fileMap.get(it[2])) }
@@ -94,7 +105,7 @@ spltnames
else {
spltnames
.splitCsv()
- .filter { fleMap.get(it[1]) != null }
+ .filter { fileMap.get(it[1]) != null }
.map { it -> tuple(it[0], fileMap.get(it[1]),'') }
.set { read }
}
@@ -103,14 +114,21 @@ if( ! read ) { error "Didn't match any input files with entries in the design fi
//
// Trim raw reads using trimgalore
//
+
process trim {
errorStrategy 'ignore'
+
input:
+
set pair_id, file(read1), file(read2) from read
+
output:
+
set pair_id, file("${pair_id}.trim.R1.fastq.gz"),file("${pair_id}.trim.R2.fastq.gz") into trimread
set pair_id, file("${pair_id}.trim.R1.fastq.gz"),file("${pair_id}.trim.R2.fastq.gz") into fusionfq
+
script:
+
"""
bash $baseDir/process_scripts/preproc_fastq/trimgalore.sh -p ${pair_id} -a ${read1} -b ${read2}
"""
@@ -126,13 +144,21 @@ process trim {
process starfusion {
errorStrategy 'ignore'
publishDir "$params.output", mode: 'copy'
+
input:
+
set pair_id, file(fq1), file(fq2) from fusionfq
+
output:
+
file("${pair_id}.starfusion.txt") into fusionout
+
when:
+
params.fusion == 'detect' && params.pairs == 'pe'
+
script:
+
"""
bash $baseDir/process_scripts/alignment/starfusion.sh -p ${pair_id} -r ${index_path} -a ${fq1} -b ${fq2} -m trinity -f
"""
@@ -143,12 +169,17 @@ process align {
publishDir "$params.output", mode: 'copy'
input:
+
set pair_id, file(fq1), file(fq2) from trimread
+
output:
+
set pair_id, file("${pair_id}.bam") into aligned
set pair_id, file("${pair_id}.bam") into aligned2
file("${pair_id}.alignerout.txt") into hsatout
+
script:
+
"""
bash $baseDir/process_scripts/alignment/rnaseqalign.sh -a $params.align -p ${pair_id} -r ${index_path} -x ${fq1} -y ${fq2}
"""
@@ -159,11 +190,16 @@ process alignqc {
publishDir "$params.output", mode: 'copy'
input:
+
set pair_id, file(bam) from aligned2
+
output:
+
file("${pair_id}.flagstat.txt") into alignstats
set file("${pair_id}_fastqc.zip"),file("${pair_id}_fastqc.html") into fastqc
+
script:
+
"""
bash $baseDir/process_scripts/alignment/bamqc.sh -p ${pair_id} -b ${bam} -y rna
"""
@@ -175,10 +211,16 @@ process parse_alignstat {
publishDir "$params.output", mode: 'copy'
input:
+
file(txt) from alignstats.toList()
file(txt) from hsatout.toList()
+
output:
+
file('alignment.summary.txt')
+
+ script:
+
"""
perl $baseDir/scripts/parse_flagstat.pl *.flagstat.txt
"""
@@ -190,11 +232,16 @@ process markdups {
publishDir "$params.output", mode: 'copy'
input:
+
set pair_id, file(sbam) from aligned
+
output:
+
set pair_id, file("${pair_id}.dedup.bam") into deduped1
set pair_id, file("${pair_id}.dedup.bam") into deduped2
+
script:
+
"""
bash $baseDir/process_scripts/alignment/markdups.sh -a $params.markdups -b $sbam -p $pair_id
"""
@@ -206,13 +253,20 @@ process markdups {
process geneabund {
errorStrategy 'ignore'
publishDir "$params.output", mode: 'copy'
+
input:
+
set pair_id, file(sbam) from deduped1
+
output:
+
file("${pair_id}.cts") into counts
file("${pair_id}.cts.summary") into ctsum
file("${pair_id}_stringtie") into strcts
file("${pair_id}.fpkm.txt") into fpkm
+
+ script:
+
"""
bash $baseDir/process_scripts/genect_rnaseq/geneabundance.sh -s $params.stranded -g ${gtf_file} -p ${pair_id} -b ${sbam}
"""
@@ -221,7 +275,9 @@ process geneabund {
process statanal {
errorStrategy 'ignore'
publishDir "$params.output", mode: 'copy'
+
input:
+
file count_file from counts.toList()
file count_sum from ctsum.toList()
file newdesign name 'design.txt'
@@ -229,29 +285,41 @@ process statanal {
file geneset name 'geneset.gmt'
file fpkm_file from fpkm.toList()
file stringtie_dir from strcts.toList()
- output:
+
+ output:
+
file "*.txt" into txtfiles
file "*.png" into psfiles
file("*.rda") into rdafiles
file("geneset.shiny.gmt") into gmtfile
+
when:
+
script:
+
"""
bash $baseDir/process_scripts/genect_rnaseq/statanal.sh -d $params.dea
"""
}
+
process gatkbam {
errorStrategy 'ignore'
publishDir "$params.output", mode: 'copy'
input:
+
set pair_id, file(rbam) from deduped2
output:
+
set file("${pair_id}.final.bam"),file("${pair_id}.final.bai") into gatkbam
+
when:
+
params.align == 'hisat' && $index_path == '/project/shared/bicf_workflow_ref/GRCh38/'
+
script:
+
"""
bash $baseDir/process_scripts/variants/gatkrunner.sh -a gatkbam_rna -b $rbam -r ${index_path}/hisat_index -p $pair_id
"""
diff --git a/workflow/scripts/check_designfile.pl b/workflow/scripts/check_designfile.pl
index 692f9ce498cf22ead33c692d9d721a9837af1055..bf941465e13a345dbe2b1d0bed9406fbd6bc4184 100755
--- a/workflow/scripts/check_designfile.pl
+++ b/workflow/scripts/check_designfile.pl
@@ -1,6 +1,9 @@
#!/usr/bin/perl -w
#check_designfile.pl
+use strict;
+use warnings;
+
my $pe = shift @ARGV;
my $dfile = shift @ARGV;
open OUT, ">design.valid.txt" or die $!;
@@ -11,7 +14,6 @@ $head =~ s/FullPathTo//g;
my @colnames = split(/\t/,$head);
my %newcols = map {$_=> 1} @colnames;
-
unless (grep(/FqR1/,@colnames)) {
die "Missing Sequence Files in Design File: FqR1\n";
}
@@ -49,16 +51,36 @@ while (my $line = <DFILE>) {
$hash{FinalBam} = $hash{SampleID}.".final.bam";
$hash{Bam} = $hash{SampleID}.".bam";
unless ($hash{SampleGroup}) {
- $j = $lnct %% 2;
+ my $j = $lnct %% 2;
$hash{SampleGroup} = $grp[$j];
}
$hash{SampleGroup} =~ s/_//g;
+ unless ($hash{FqR1} =~ m/.fastq.gz/) {
+ my $name = $hash{FqR1};
+ $name =~ s/.f.*/.fastq.gz/;
+ unless ($hash{FqR1} eq $name) {
+ $hash{FqR1} = $name;
+ unless (-e ($name)) {
+ die "Unable to find fastq read 1\n${name}\n";
+ }
+ }
+ }
+ $hash{FqR2} = 'na' unless ($hash{FqR2});
+ unless ($hash{FqR2} eq 'na') {
+ my $name = $hash{FqR2};
+ $name =~ s/.f.*/.fastq.gz/;
+ unless ($hash{FqR2} eq $name) {
+ $hash{FqR2} = $name;
+ unless (-e ($name)) {
+ die "Unable to find fastq read 2\n${name}\n";
+ }
+ }
+ }
my @line;
- foreach $f (@cols) {
+ foreach my $f (@cols) {
push @line, $hash{$f};
}
print OUT join("\t",@line),"\n";
- $hash{FqR2} = 'na' unless ($hash{FqR2});
print join(",",$hash{SampleID},$hash{FqR1},$hash{FqR2}),"\n";
$lnct ++;
}