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rnaseq_pipeline.pl
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rnaseq_pipeline.pl
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#!/usr/bin/perl
use strict;
use Getopt::Long qw(GetOptions);
use FindBin qw($Bin);
use lib "$Bin/lib";
use Schedule;
use Cluster;
### takes a list of directories containing fastq.gz files
### 1) makes directories for each sample
### 2) soft lins to all fastq.gz files in directory
### ex. input list $file
### LIB_ID SAMPLE_ID RUN_id /ifs/archive/GCL/hiseq/FASTQ/LIZ_0096_AB07L5ABXX/Project_2144/Sample_14028 PE
### LIB_ID SAMPLE_ID RUN_id /ifs/archive/GCL/hiseq/FASTQ/LIZ_0096_AB07L5ABXX/Project_2144/Sample_14278 PE
### LIB_ID SAMPLE_ID RUN_id /ifs/archive/GCL/hiseq/FASTQ/LIZ_0096_AB07L5ABXX/Project_2144/Sample_14314 PE
### LIB_ID SAMPLE_ID RUN_id /ifs/archive/GCL/hiseq/FASTQ/LIZ_0096_AB07L5ABXX/Project_2144/Sample_14348 SE
### LIB_ID SAMPLE_ID RUN_id /ifs/archive/GCL/hiseq/FASTQ/LIZ_0096_AB07L5ABXX/Project_2144/Sample_14512 SE
### etc.
### config file: list of programs
### PROGRAM_NAME PATH_TO_PROGRAM
### ex.
### STAR /opt/common/star/STAR_2.3.0e
### PICARD /opt/common/picard/picard-tools-1.104
### NOTE: MUST GIVE FULL PATH TO CONFIG FILE
### NOTE2: FOR FUSION, IF A SAMPLE CONSISTS OF PE AND SE READS e.g. HAS MULTIPLE RUNS, AND SOME ARE PE AND OTHERS ARE SE
### THIS WILL CAUSE FUSIONS TO NOT WORK BECAUSE OF UNEVEN READ FILES CAUSE DURING CAT OF ALL READS
my ($map, $pre, $config, $request, $help, $species, $cufflinks, $dexseq, $htseq, $chimerascan, $samplekey, $comparisons, $deseq, $star_fusion, $mapsplice, $defuse, $fusioncatcher, $detectFusions, $allfusions, $tophat, $star, $pass1, $lncrna, $lincrna_BROAD, $output, $strand, $r1adaptor, $r2adaptor, $scheduler, $rsem, $kallisto, $express, $standard_gene, $clustering_only, $differential_gene, $standard_transcript, $differential_transcript, $alignment_only, $resume_deseq);
$pre = 'TEMP';
$output = "results";
my $priority_project = "ngs";
my $priority_group = "Pipeline";
my $rseqc_batch_size = 10;
my $uID = `/usr/bin/id -u -n`;
chomp $uID;
my $svnRev = `svn info $Bin | grep Revision | cut -d " " -f 2`;
chomp $svnRev;
my $email = "$uID\@cbio.mskcc.org";
my $rsync = "/juno/res/bic/$uID";
GetOptions ('map=s' => \$map,
'pre=s' => \$pre,
'config=s' => \$config,
'request=s' => \$request,
'samplekey=s' => \$samplekey,
#'comparisons=s' => \$comparisons,
'h|help' => \$help,
'star' => \$star,
'pass1' => \$pass1,
'tophat|tophat2' => \$tophat,
'cufflinks' => \$cufflinks,
'dexseq' => \$dexseq,
'htseq' => \$htseq,
'deseq' => \$deseq,
'chimerascan' => \$chimerascan,
'star_fusion' => \$star_fusion,
'mapsplice' => \$mapsplice,
'defuse' => \$defuse,
'fusioncatcher' => \$fusioncatcher,
'allfusions' => \$allfusions,
'kallisto' => \$kallisto,
'rsem' => \$rsem,
'express' => \$express,
'species=s' => \$species,
'strand=s' => \$strand,
'standard_gene' => \$standard_gene,
'differential_gene|diff_gene' => \$differential_gene,
'clustering_only' => \$clustering_only,
'standard_transcript|standard_trans' => \$standard_transcript,
'differential_transcript|diff_trans' => \$differential_transcript,
'lncrna' => \$lncrna,
'output|out|o=s' => \$output,
'rsync=s' => \$rsync,
'email' => \$email,
'r1adaptor=s' => \$r1adaptor,
'r2adaptor=s' => \$r2adaptor,
'priority_project=s' => \$priority_project,
'priority_group=s' => \$priority_group,
'lincrna_BROAD' => \$lincrna_BROAD,
'alignment_only' =>\$alignment_only,
'resume_deseq' =>\$resume_deseq) or exit(1);
if(!$map || !$species || !$strand || !$config || !$request || $help){
print <<HELP;
USAGE: rnaseq_pipeline.pl -map MAP -species SPECIES -strand STRAND -config CONFIG -pre PRE -samplekey SAMPLEKEY -comparisons COMPARISONS -request REQUEST
* MAP: file listing sample mapping information for processing (REQUIRED)
* SPECIES: only hg19, mouse (mm10; default) and human-mouse hybrid (hybrid), zebrafish (z11), fly (dm6) currently supported (REQUIRED)
* STRAND: library strand; valid options are none, forward, reverse (REQUIRED)
* CONFIG: file listing paths to programs needed for pipeline; full path to config file needed (REQUIRED)
* REQUEST: file containing all request information including PI, Investigator and ProjectID (REQUIRED)
* SCHEDULER: currently support for SGE, LUNA and JUNO (REQUIRED)
* PRE: output prefix (default: TEMP)
* STANDARD_GENE: standard analysis - star alignment, htseq gene count
* DIFFERENTIAL_GENE: differential gene analysis - star alignment, htseq gene count, deseq, counts normalization, clustering, and gsa
* STANDARD_TRANSCRIPT: standard analysis - rsem transcript counts
* DIFFERENTIAL_TRANSCRIPT: differential transcript analysis - rsem transcript counts, deseq, counts normalization, and clustering
* SAMPLEKEY: tab-delimited file listing sampleName in column A and condition in column B (if -deseq, REQUIRED)
* COMPARISONS: tab-delimited file listing the conditions to compare in columns A/B (if -deseq, REQUIRED)
* EMAIL: email to send notication of finished final job of pipeline (default: $uID\@cbio.mskcc.org)
* RSYNC: path to rsync data for archive (default: /ifs/res/$uID)
* R1ADAPTOR/R2ADAPTOR: if provided, will trim adaptor sequences; NOTE: if provided for only one end, will also assign it to the other end
* ALIGNERS SUPPORTED: star (-star), defaults to 2pass method unless -pass1 specified; tophat2 (-tophat); if no aligner specifed, will default to STAR
* ANALYSES SUPPORTED: cufflinks (-cufflinks); htseq (-htseq); dexseq (-dexseq); deseq (-deseq; must specify samplekey and comparisons); fusion callers chimerascan (-chimerascan), rna star (-star_fusion), mapsplice (-mapsplice), defuse (-defuse), fusioncatcher (-fusioncatcher); -allfusions will run all supported fusion detection programs; transcript analysis using express (-express), kallisto (-kallisto) and rsem (-rsem), and all (-transcript)
* PRIORITY_PROJECT: sge notion of priority assigned to projects (default: ngs)
* PRIORITY_GROUP: lsf notion of priority assigned to groups (default: Pipeline)
* OUTPUT: output results directory (default: results)
* OPTIONS: lncRNA analysis (-lncrna) runs all analyses based on lncRNA GTF (hg19 only); -resume_deseq prevents full rerun of all previously save DESeq analyses
HELP
exit;
}
if($ENV{'LSF_ENVDIR'} eq "/common/lsf/conf"){
$scheduler = 'luna';
}
elsif($ENV{'LSF_ENVDIR'} eq "/common/juno/OS7/conf" or $ENV{'LSF_ENVDIR'} eq "/admin/lsfjuno/lsf/conf"){
$scheduler = 'juno';
}
elsif($ENV{'SGE_ROOT'} ne ""){
$scheduler = 'sge';
}
else{
die "unrecognized scheduler, valid scheduler [sge, luna, juno]";
}
if($pre =~ /^\d+/){
$pre = "s_$pre";
}
my $curDir = `pwd`;
chomp $curDir;
my $cd = $curDir;
$cd =~ s/\//_/g;
if($output !~ /^\//){
$output = "$curDir/$output";
}
if($species !~ /human|hg19|mouse|mm9|mm10|hybrid|zebrafish|zv9|zv10|z11|dm6|fly|WBcel235|sacCer3|rat|Rnor6|rn6/i){
die "Species must be human (hg19), mouse (mm9/mm10), human-mouse hybrid (hybrid), fly (dm6), C.elegans (WBcel235), yeast (sacCer3) or zebrafish (zv9/zv10/z11)";
}
if($r1adaptor){
if(!$r2adaptor){
$r2adaptor = $r1adaptor;
}
}
else{
if($r2adaptor){
$r1adaptor = $r2adaptor;
}
}
### dumb reconstruction of command line
my $commandLine = "$Bin/rnaseq_pipeline.pl";
if($pre){
$commandLine .= " -pre $pre";
}
if($map){
$commandLine .= " -map $map";
}
if($config){
$commandLine .= " -config $config";
}
if($request){
$commandLine .= " -request $request";
}
if($strand){
$commandLine .= " -strand $strand";
}
if($samplekey){
$commandLine .= " -samplekey $samplekey";
}
#if($comparisons){
# $commandLine .= " -comparisons $comparisons";
#}
if($scheduler){
$commandLine .= " -scheduler $scheduler";
}
if($r1adaptor){
$commandLine .= " -r1adaptor $r1adaptor";
}
if($r2adaptor){
$commandLine .= " -r2adaptor $r2adaptor";
}
if($standard_gene){
$commandLine .= " -standard_gene";
}
if($standard_transcript){
$commandLine .= " -standard_transcript";
}
if($star){
$commandLine .= " -star";
}
if($pass1){
$commandLine .= " -pass1";
}
if($tophat){
$commandLine .= " -tophat";
}
if($cufflinks){
$commandLine .= " -cufflinks";
}
if($dexseq){
$commandLine .= " -dexseq";
}
if($htseq){
$commandLine .= " -htseq";
}
if($deseq){
$commandLine .= " -deseq";
}
if($resume_deseq){
$commandLine .= " -resume_deseq";
}
if($chimerascan){
$commandLine .= " -chimerascan";
}
if($star_fusion){
$commandLine .= " -star_fusion";
}
if($mapsplice){
$commandLine .= " -mapsplice";
}
if($defuse){
$commandLine .= " -defuse";
}
if($fusioncatcher){
$commandLine .= " -fusioncatcher";
}
if($allfusions){
$commandLine .= " -allfusions";
}
if($kallisto){
$commandLine .= " -kallisto";
}
if($rsem){
$commandLine .= " -rsem";
}
if($express){
$commandLine .= " -express";
}
if($species){
$commandLine .= " -species $species";
}
if($lncrna){
$commandLine .= " -lncrna";
}
if($rsync){
$commandLine .= " -rsync $rsync";
}
if($email){
$commandLine .= " -email $email";
}
$commandLine.= " -priority_project $priority_project";
$commandLine .= " -priority_group $priority_group";
my $numArgs = $#ARGV + 1;
foreach my $argnum (0 .. $#ARGV) {
$commandLine .= " $ARGV[$argnum]";
}
if($standard_gene){
$star = 1;
$htseq = 1;
}
if($differential_gene){
$star = 1;
$htseq = 1;
$deseq = 1;
}
if($clustering_only){
$star = 1;
$htseq = 1;
}
if($standard_transcript){
$rsem = 1;
}
if($differential_transcript){
$rsem = 1;
$deseq = 1;
}
if($star && !$htseq && !$dexseq){
$htseq = 1;
}
if($allfusions){
$chimerascan = 1;
$star_fusion = 1;
$mapsplice = 1;
$defuse = 1;
$fusioncatcher = 1;
}
if($chimerascan || $star_fusion || $mapsplice || $defuse || $fusioncatcher){
$detectFusions = 1;
}
if($deseq || $dexseq || $htseq || $cufflinks){
if(!$star && !$tophat && !$rsem){
$star = 1;
my @currentTime = &getTime();
print LOG "$currentTime[2]:$currentTime[1]:$currentTime[0], $currentTime[5]\/$currentTime[4]\/$currentTime[3]\tNO ALIGNER SPECIFIED SO DEFAULTING TO USING STAR\n";
}
}
`/bin/mkdir -m 775 -p $output`;
## if no sample key specified, check for any that exist and if they do,
## read them to check for samples that should be excluded
my @sample_keys = $samplekey;
my @included_samples = ();
if($deseq || $clustering_only){
if(!$samplekey){
@sample_keys = glob("*sample_key*.txt");
foreach my $fl (@sample_keys){
print "$fl\n";
}
}
my $keyCount = scalar @sample_keys;
if($keyCount == 0 && $deseq){
die "No key file found. Can not run DESeq.\n";
}
foreach my $sample_key (@sample_keys){
print "Checking for exclusions in $sample_key\n";
open(SK, "$sample_key") or die "Can't open sample key $sample_key $!";
while(<SK>){
chomp;
my @samp_group = split(/\s+/, $_);
if(uc($samp_group[1]) eq "_EXCLUDE_"){
print "Excluding $samp_group[0]\t$samp_group[1] from these comparisons\n";
} else {
push(@included_samples, $samp_group[0]);
}
}
close SK;
}
}
my %mapping_samples = ();
my %ism_samples = ();
open(MA, "$map") or die "Can't open mapping file $map $!";
while(<MA>){
chomp;
my @data = split(/\s+/, $_);
if($data[0] eq "V1"){
die "ERROR: Looks like key file has R header included...?\n";
}
if(scalar(@included_samples) > 0){
my %incl = map { $_, 1 } @included_samples;
if( !$incl{ $data[1] } ){
print "EXCLUDING sample $data[1] from ALL analysis\n";
next;
}
}
$mapping_samples{$data[1]} = 1;
## this check messes things up because of the way permissions are set up on /igo.
## the permissions on the sample directory causes this 'die' to be executed, but the
## fastqs in that directory can still be read
if(!-d $data[3]){
die "$data[3] does not exist\n";
}
## keep track of PE/SE for InsertSizeMetrics
if($data[4] eq "PE"){
$ism_samples{$data[1]} = 1;
}
`/bin/cp $map $output/`;
}
close MA;
my $BOWTIE2 = '';
my $CUFFLINKS = '';
my $HTSEQ = '';
my $DEXSEQ = '';
my $EXPRESS = '';
my $KALLISTO = '';
my $PICARD = '';
my $CHIMERASCAN = '';
my $MAPSPLICE = '';
my $STAR = '';
my $DEFUSE = '';
my $FUSIONCATCHER = '';
my $TOPHAT = '';
my $PYTHON = '';
my $JAVA = '';
my $PERL = '';
my $R = '';
my $SINGULARITY_R = '';
my $RSEM = '';
my $CUTADAPT = '';
my $STAR_FUSION = '';
my $SINGULARITY = '';
my $CIBERSORT = '';
my $singularityParams = '';
my $singularityBind = '';
my $singularityenv_prepend_path = '';
my $Rlibs = '';
my %samp_libs_run = ();
my $slr_count = 0;
my %samp_pair = ();
open(LOG, ">$cd\_rnaseq_pipeline.log") or die "can't write to output log";
my @currentTime = &getTime();
print LOG "$currentTime[2]:$currentTime[1]:$currentTime[0], $currentTime[5]\/$currentTime[4]\/$currentTime[3]\tSTARTING RNASEQ PIPELINE FOR $pre\n";
print LOG "$currentTime[2]:$currentTime[1]:$currentTime[0], $currentTime[5]\/$currentTime[4]\/$currentTime[3]\tCOMMAND LINE: $commandLine\n";
### Check that all programs are available
&verifyConfig($config);
my $GTF = '';
my $DEXSEQ_GTF = '';
my $CHIMERASCAN_INDEX = '';
my $geneNameConversion = '';
my $starDB = '';
my $chrSplits = '';
my $BOWTIE_INDEX = '';
my $BOWTIE2_INDEX = '';
my $TRANS_FASTA_DEDUP = '';
my $TRANS_INDEX = '';
my $TRANS_INDEX_DEDUP = '';
my $REF_SEQ = '';
my $RIBOSOMAL_INTERVALS='';
my $REF_FLAT = '';
my $KALLISTO_INDEX = '';
my $STAR_FUSION_GENOME_LIB = '';
my $CHIMERASCAN_FP_FILTER = '';
my $RSEM_DB = '';
my $QC_BED = '';
my $GMT_DIR = '';
my $STAR_MAX_MEM = 100;
if($species =~ /human|^hg19$/i){
$species = 'hg19';
$REF_SEQ = '/juno/depot/assemblies/H.sapiens/hg19/hg19.fasta';
$DEXSEQ_GTF = "$Bin/data/human/gencode.v18.annotation_dexseq.gtf";
$QC_BED = "$Bin/data/hg19/hg19_GENCODE_GENE_V19_comprehensive.bed";
$CHIMERASCAN_INDEX = '/juno/depot/assemblies/H.sapiens/hg19/index/chimerascan/0.4.5a';
$BOWTIE_INDEX = '/juno/depot/assemblies/H.sapiens/hg19/index/bowtie/1.0.0/hg19_bowtie';
$BOWTIE2_INDEX = '/juno/depot/assemblies/H.sapiens/hg19/index/bowtie/2.2.4/hg19_bowtie2';
$chrSplits = '/juno/depot/assemblies/H.sapiens/hg19/chromosomes';
$RIBOSOMAL_INTERVALS = "$Bin/data/hg19/ribosomal_hg19.interval_file";
$REF_FLAT = "$Bin/data/hg19/refFlat__hg19.txt.gz";
$TRANS_INDEX = '/juno/depot/assemblies/H.sapiens/hg19/index/bowtie/2.2.4/transcriptome/gencode/v18/gencode.v18.annotation';
$TRANS_INDEX_DEDUP = '/juno/depot/assemblies/H.sapiens/hg19/index/bowtie/2.2.4/transcriptome/gencode/v18/deduplicated/gencode.v18.annotation.dedup';
$TRANS_FASTA_DEDUP = '/juno/depot/assemblies/H.sapiens/hg19/index/bowtie/2.2.4/transcriptome/gencode/v18/deduplicated/gencode.v18.annotation.dedup.fa';
$geneNameConversion = "$Bin/data/human/gencode18IDToGeneName.txt";
$KALLISTO_INDEX = '/juno/depot/assemblies/H.sapiens/hg19/index/kallisto/v0.42.1/gencode/v18/gencode.v18.annotation.gtf.fasta.idx';
$RSEM_DB = '/opt/common/CentOS_6/rsem/RSEM-1.2.25/data/hg19/star/hg19_v19_gencode';
$STAR_FUSION_GENOME_LIB = "$STAR_FUSION/Hg19_CTAT_resource_lib";
$CHIMERASCAN_FP_FILTER = "$Bin/data/hg19/hg19_bodymap_false_positive_chimeras.txt";
$GMT_DIR = "$Bin/data/human/MSigDB/v7.1";
if($lncrna){
$GTF = "$Bin/data/human/lncipedia.gtf";
$starDB = '/juno/depot/assemblies/H.sapiens/hg19/index/star/2.3.0e_r291/LNCipedia';
} else {
$GTF = "$Bin/data/human/gencode.v18.annotation.gtf";
if($r1adaptor){
$starDB = '/juno/depot/assemblies/H.sapiens/hg19/index/star/2.4.1d/gencode/v18/overhang49';
}
else{
$starDB = '/juno/depot/assemblies/H.sapiens/hg19/index/star/2.4.1d/gencode/v18/overhang74';
}
}
$STAR_MAX_MEM = 35;
}
elsif($species =~ /mouse|^mm10$/i){
$species = 'mm10';
$REF_SEQ = '/juno/depot/assemblies/M.musculus/mm10/mm10.fasta';
$QC_BED = "$Bin/data/mm10/mm10_GENCODE_VM9_basic.bed";
$GTF = "$Bin/data/mm10/gencode.vM8.annotation.gtf";
#$DEXSEQ_GTF = "$Bin/data/Mus_musculus.GRCm38.80_canonical_chromosomes.dexseq.gtf";
$geneNameConversion = "$Bin/data/mm10/gencodeM8IDToGeneName.txt";
$BOWTIE_INDEX = '/juno/depot/assemblies/M.musculus/mm10/index/bowtie/1.1.1/mm10_bowtie';
$BOWTIE2_INDEX = '/juno/depot/assemblies/M.musculus/mm10/index/bowtie/2.2.4/mm10_bowtie2';
$chrSplits = '/juno/depot/assemblies/M.musculus/mm10/chromosomes';
$TRANS_INDEX = '/juno/depot/assemblies/M.musculus/mm10/index/bowtie/2.2.4/transcriptome/gencode/vM8/gencode.vM8.annotation';
$TRANS_INDEX_DEDUP = '/juno/depot/assemblies/M.musculus/mm10/index/bowtie/2.2.4/transcriptome/gencode/vM8/deduplicated/gencode.vM8.annotation.dedup';
$TRANS_FASTA_DEDUP = '/juno/depot/assemblies/M.musculus/mm10/index/bowtie/2.2.4/transcriptome/gencode/vM8/deduplicated/gencode.vM8.annotation.dedup.fa';
$RIBOSOMAL_INTERVALS = "$Bin/data/mm10/ribosomal_mm10.interval_file";
$REF_FLAT = "$Bin/data/mm10/refFlat__mm10.txt.gz";
$KALLISTO_INDEX = '/juno/depot/assemblies/M.musculus/mm10/index/kallisto/v0.42.1/gencode/vM8/gencode.vM8.annotation.gtf.fasta.idx';
$RSEM_DB = '/opt/common/CentOS_6/rsem/RSEM-1.2.25/data/GRCm38/star/GRCm38_vM8_gencode';
$GMT_DIR = "$Bin/data/mouse/MSigDB/v7.1";
if($r1adaptor){
$starDB = '/juno/depot/assemblies/M.musculus/mm10/index/star/2.4.1d/gencode/vM8/overhang49';
}
else{
$starDB = '/juno/depot/assemblies/M.musculus/mm10/index/star/2.4.1d/gencode/vM8/overhang74';
}
$STAR_MAX_MEM = 30;
}
elsif($species =~ /^mm9$/i){
$species = 'mm9';
$REF_SEQ = '/juno/depot/assemblies/M.musculus/mm9/mm9.fasta';
$GTF = "$Bin/data/mm9/Mus_musculus.NCBIM37.67_ENSEMBL.gtf";
$DEXSEQ_GTF = "$Bin/data/mm9/Mus_musculus.NCBIM37.67_ENSEMBL.dexseq.gtf";
$geneNameConversion = "$Bin/data/mm9/mm9Ensembl67IDToGeneName.txt";
$BOWTIE_INDEX = '/juno/depot/assemblies/M.musculus/mm9/index/bowtie/1.0.0/mm9_bowtie';
$BOWTIE2_INDEX = '/juno/depot/assemblies/M.musculus/mm9/index/bowtie/2.1.0/mm9_bowtie2';
$chrSplits = '/juno/depot/assemblies/M.musculus/mm9/chromosomes';
$TRANS_INDEX = '/juno/depot/assemblies/M.musculus/mm9/index/bowtie/2.1.0/transcriptome/ensembl/vTBD/ensembl';
$TRANS_INDEX_DEDUP = '';
$TRANS_FASTA_DEDUP = '';
$RIBOSOMAL_INTERVALS = "$Bin/data/mm9/ribosomal_MM9_assemblies.interval_file";
$REF_FLAT = "$Bin/data/mm9/refFlat__mm9.txt.gz";
$KALLISTO_INDEX = '';
$RSEM_DB = '/opt/common/CentOS_6/rsem/RSEM-1.2.25/data/mm9/star/mm9_vm1_gencode';
$GMT_DIR = "$Bin/data/mouse/MSigDB/v7.1";
if($r1adaptor){
$starDB = '/juno/depot/assemblies/M.musculus/mm9/index/star/2.4.1d/ensembl/v67/overhang49';
}
else{
$starDB = '/juno/depot/assemblies/M.musculus/mm9/index/star/2.4.1d/ensembl/v67/overhang74';
}
$STAR_MAX_MEM = 30;
}
elsif($species =~ /human-mouse|mouse-human|hybrid|^hg19_mm10$/i){
$species = 'hybrid';
$REF_SEQ = '/juno/depot/assemblies/hybrids/H.sapiens_M.musculus/hg19_mm10/hg19_mm10.fasta';
$GTF = "$Bin/data/human/gencode.v18.annotation.gtf";
$DEXSEQ_GTF = "$Bin/data/human/gencode.v18.annotation_dexseq.gtf";
$geneNameConversion = "$Bin/data/human/gencode18IDToGeneName.txt";
$RIBOSOMAL_INTERVALS = "$Bin/data/hg19_mm10/ribosomal_hg19_mm10.interval_file";
$REF_FLAT = "$Bin/data/hg19/refFlat__hg19.txt.gz";
$RSEM_DB = '/juno/depot/assemblies/hybrids/H.sapiens_M.musculus/hg19_mm10/index/rsem/1.2.25/gencode/v18/hg19_mm10';
$BOWTIE_INDEX = '/juno/depot/assemblies/hybrids/H.sapiens_M.musculus/hg19_mm10/index/bowtie/1.1.1/hg19_mm10_bowtie';
$BOWTIE2_INDEX = '/juno/depot/assemblies/hybrids/H.sapiens_M.musculus/hg19_mm10/index/bowtie/2.2.4/hg19_mm10_bowtie2';
$KALLISTO_INDEX = '/juno/depot/assemblies/hybrids/H.sapiens_M.musculus/hg19_mm10/index/kallisto/v0.42.1/gencode/v18/gencode.v18.annotation.gtf.fasta.idx';
$STAR_FUSION_GENOME_LIB = "$STAR_FUSION/Hg19_CTAT_resource_lib";
$CHIMERASCAN_INDEX = "/juno/depot/assemblies/hybrids/H.sapiens_M.musculus/hg19_mm10/index/chimerascan/0.4.5a";
$CHIMERASCAN_FP_FILTER = "$Bin/data/hg19/hg19_bodymap_false_positive_chimeras.txt";
$chrSplits = '/juno/depot/assemblies/hybrids/H.sapiens_M.musculus/hg19_mm10/chromosomes';
$QC_BED = "$Bin/data/hg19/hg19_GENCODE_GENE_V19_comprehensive.bed";
$GMT_DIR = "$Bin/data/human/MSigDB/v7.1";
if($r1adaptor){
$starDB = '/juno/depot/assemblies/hybrids/H.sapiens_M.musculus/hg19_mm10/index/star/2.4.1d/gencode/v18/overhang49';
}
else{
$starDB = '/juno/depot/assemblies/hybrids/H.sapiens_M.musculus/hg19_mm10/index/star/2.4.1d/gencode/v18/overhang74';
}
$STAR_MAX_MEM = 60;
}
elsif($species =~ /rat|^Rn6$/i){
$species = 'Rn6';
$REF_SEQ = '/juno/depot/assemblies/R.norvegicus/Rnor_6.0/Rnor_6.0.fasta';
$GTF = "$Bin/data/rn6/Rattus_norvegicus.Rnor_6.0.92.gtf";
$geneNameConversion = "$Bin/data/rn6/rn6Ensembl92IDToGeneName.txt";
$BOWTIE_INDEX = '/juno/depot/assemblies/R.norvegicus/Rnor_6.0/index/bowtie/1.1.1/Rnor_6.0_bowtie';
$BOWTIE2_INDEX = '/juno/depot/assemblies/R.norvegicus/Rnor_6.0/index/bowtie/2.2.4/Rnor_6.0_bowtie2';
$chrSplits = '/juno/depot/assemblies/R.norvegicus/Rnor_6.0/chromosomes';
$TRANS_INDEX = '/juno/depot/assemblies/R.norvegicus/Rnor_6.0/index/bowtie/2.2.4/transcriptome/ensembl/v92/Rattus_norvegicus.Rnor_6.0.92';
$TRANS_INDEX_DEDUP = '/juno/depot/assemblies/R.norvegicus/Rnor_6.0/index/bowtie/2.2.4/transcriptome/ensembl/v92/deduplicated/Rattus_norvegicus.Rnor_6.0.92.gtf.dedup';
$TRANS_FASTA_DEDUP = '/juno/depot/assemblies/R.norvegicus/Rnor_6.0/index/bowtie/2.2.4/transcriptome/ensembl/v92/deduplicated/Rattus_norvegicus.Rnor_6.0.92.gtf.dedup.fa';
$RIBOSOMAL_INTERVALS = "$Bin/data/rn6/ribosomal_Rnor6.interval_file";
$REF_FLAT = "$Bin/data/rn6/refFlat__rn6.txt";
$KALLISTO_INDEX = '';
$RSEM_DB = '';
if($r1adaptor){
$starDB = '/juno/depot/assemblies/R.norvegicus/Rnor_6.0/index/star/2.4.1d/ensembl/v92/overhang49';
}
else{
$starDB = '/juno/depot/assemblies/R.norvegicus/Rnor_6.0/index/star/2.4.1d/ensembl/v92/overhang74';
}
$STAR_MAX_MEM = 30;
}
elsif($species =~ /zebrafish|z11|GRCz11/i){
$species = 'z11';
$REF_SEQ = '/juno/depot/assemblies/D.rerio/GRCz11/GRCz11.fasta';
$GTF = '/juno/depot/assemblies/D.rerio/GRCz11/Danio_rerio.GRCz11.97.gtf';
if($r1adaptor){
$starDB = '/juno/depot/assemblies/D.rerio/GRCz11/index/star/2.4.1d/ensembl/v97/overhang49';
} else {
$starDB = '/juno/depot/assemblies/D.rerio/GRCz11/index/star/2.4.1d/ensembl/v97/overhang74';
}
$chrSplits = '/juno/depot/assemblies/D.rerio/GRCz11/chromosomes';
$geneNameConversion = "$Bin/data/GRCz11/IDToGeneName.txt";
$TRANS_INDEX = '';
$TRANS_INDEX_DEDUP = '';
$TRANS_FASTA_DEDUP = '';
$RIBOSOMAL_INTERVALS = "$Bin/data/GRCz11/ribosomal_GRCz11.97.interval_file";
$REF_FLAT = "$Bin/data/GRCz11/refFlat__GRCz11.txt.gz";
$RSEM_DB = "";
$KALLISTO_INDEX = "";
$QC_BED = "$Bin/data/GRCz11/Danio_rerio.GRCz11.97.bed";
$STAR_MAX_MEM = 30;
}
elsif($species =~ /zv10/i){
$species = 'zv10';
$REF_SEQ = '/juno/depot/assemblies/D.rerio/GRCz10/GRCz10.fasta';
$GTF = "$Bin/data/zv10/GRCz10.gtf";
if($r1adaptor){
$starDB = '/juno/depot/assemblies/D.rerio/GRCz10/index/star/2.5.0a/ensembl/v83/overhang49';
} else {
$starDB = '/juno/depot/assemblies/D.rerio/GRCz10/index/star/2.5.0a/ensembl/v83/overhang74';
}
$chrSplits = '/juno/depot/assemblies/D.rerio/GRCz10/chromosomes';
$geneNameConversion = "$Bin/data/zv10/zv10EnsemblIDtoGeneName.txt";
$TRANS_INDEX = '';
$TRANS_INDEX_DEDUP = '';
$TRANS_FASTA_DEDUP = '';
$RIBOSOMAL_INTERVALS = "$Bin/data/zv10/ribosomal_zv10.interval_file";
$REF_FLAT = "$Bin/data/zv10/refFlat__zv10.txt.gz";
$RSEM_DB = '/opt/common/CentOS_6/rsem/RSEM-1.2.25/data/zv10/star/zv10_v83_ensembl';
$KALLISTO_INDEX = '';
$QC_BED = "$Bin/data/zv10/zv10_ENSEMBL_20180417.bed";
$STAR_MAX_MEM = 30;
}
elsif($species =~ /zv9/i){
$species = 'zv9';
$REF_SEQ = '/juno/depot/assemblies/D.rerio/zv9/zv9.fasta';
$GTF = "$Bin/data/zv9/zv9.gtf";
if($r1adaptor){
$starDB = '/juno/depot/assemblies/D.rerio/zv9/index/star/2.4.1d/ensembl/v79/overhang49';
} else {
$starDB = '/juno/depot/assemblies/D.rerio/zv9/index/star/2.4.1d/ensembl/v79/overhang74';
}
$chrSplits = '/juno/depot/assemblies/D.rerio/zv9/chromosomes';
$geneNameConversion = "Bin/data/zv9/zv9EnsemblIDtoGeneName.txt";
$TRANS_INDEX = '';
$TRANS_INDEX_DEDUP = '';
$TRANS_FASTA_DEDUP = '';
$RIBOSOMAL_INTERVALS = '';
$REF_FLAT = '';
$KALLISTO_INDEX = '';
$STAR_MAX_MEM = 30;
}
elsif($species =~ /fly|dmel6|dm6/i){
$species = 'dm6';
$REF_SEQ = '/juno/depot/assemblies/D.melanogaster/dm6/dm6.fasta';
$GTF = "/juno/depot/assemblies/D.melanogaster/dm6/dmel-all-r6.33.gtf";
$QC_BED = "/juno/depot/assemblies/D.melanogaster/dm6/dm6_refseq_all.bed";
$DEXSEQ_GTF = "";
$geneNameConversion = "/juno/depot/assemblies/D.melanogaster/dm6/IDToGeneName.txt";
$BOWTIE_INDEX = '/juno/depot/assemblies/D.melanogaster/dm6/index/bowtie/1.1.1/dm6_bowtie';
$BOWTIE2_INDEX = '/juno/depot/assemblies/D.melanogaster/dm6/index/bowtie/2.2.4/dm6_bowtie2';
$RSEM_DB = '/juno/depot/assemblies/D.melanogaster/dm6/index/rsem/1.3.3/flybase/6.33/star/dm6';
$chrSplits = '/juno/depot/assemblies/D.melanogaster/dm6/chromosomes';
$TRANS_INDEX = '';
$TRANS_INDEX_DEDUP = '';
$TRANS_FASTA_DEDUP = '';
$RIBOSOMAL_INTERVALS = '/juno/depot/assemblies/D.melanogaster/dm6/ribosomal_dm6.interval_file';
$REF_FLAT = '/juno/depot/assemblies/D.melanogaster/dm6/dm6_refFlat.txt';
$KALLISTO_INDEX = '';
if($r1adaptor){
$starDB = '/juno/depot/assemblies/D.melanogaster/dm6/index/star/2.4.1d/flybase/6.33/overhang49';
}
else{
$starDB = '/juno/depot/assemblies/D.melanogaster/dm6/index/star/2.4.1d/flybase/6.33/overhang74';
}
$STAR_MAX_MEM = 30;
}
elsif($species =~ /WBcel235/i){
$species = 'WBcel235';
$REF_SEQ = '/juno/depot/assemblies/custom/C.elegans/WBcel235/WBcel235.fasta';
$GTF = "$Bin/data/WBcel235/ensembl_WBcel235.gtf";
$DEXSEQ_GTF = '';
$CHIMERASCAN_INDEX = '';
$BOWTIE_INDEX = '/juno/depot/assemblies/custom/C.elegans/WBcel235/index/bowtie/1.1.1/WBcel235_bowtie';
$BOWTIE2_INDEX = '/juno/depot/assemblies/custom/C.elegans/WBcel235/index/bowtie/2.2.4/WBcel235_bowtie2';
$chrSplits = '/juno/depot/assemblies/custom/C.elegans/WBcel235/chromosomes';
$RIBOSOMAL_INTERVALS = "$Bin/data/WBcel235/ribosomal_WBcel235.interval_file";
$REF_FLAT = "$Bin/data/WBcel235/refFlat__ce11.txt.gz";
$TRANS_INDEX = '/juno/depot/assemblies/custom/C.elegans/WBcel235/index/bowtie/2.2.4/transcriptome/ensembl/v20151123/genes';
$TRANS_INDEX_DEDUP = '';
$TRANS_FASTA_DEDUP = '';
$geneNameConversion = "$Bin/data/WBcel235/WBcel235EnsemblIDToGeneName.txt";
$KALLISTO_INDEX = '/juno/depot/assemblies/custom/C.elegans/WBcel235/index/kallisto/v0.42.1/ensembl/v20151123/genes.gtf.fasta.idx';
$RSEM_DB = '/juno/depot/assemblies/custom/C.elegans/WBcel235/index/rsem/1.2.25/star/2.4.1d/WBcel235_ensembl';
$STAR_FUSION_GENOME_LIB = '';
$CHIMERASCAN_FP_FILTER = '';
if($r1adaptor){
$starDB = '/juno/depot/assemblies/custom/C.elegans/WBcel235/index/star/2.4.1d/ensembl/v20151123/overhang49';
}
else{
$starDB = '/juno/depot/assemblies/custom/C.elegans/WBcel235/index/star/2.4.1d/ensembl/v20151123/overhang74';
}
$STAR_MAX_MEM = 30;
}
elsif($species =~ /yeast|s288c|R64|sacCer3/i){
$species = 'S288C_R64';
$REF_SEQ = '/juno/depot/assemblies/S.cerevisiae/S288C_R64/S288C_R64.fasta';
$GTF = '/juno/depot/annotation/S.cerevisiae/ensembl/v94/Saccharomyces_cerevisiae.R64-1-1.94.gtf';
$QC_BED = "$Bin/data/S288C_R64/Saccharomyces_cerevisiae.R64-1-1.94.bed";
$DEXSEQ_GTF = '';
$CHIMERASCAN_INDEX = '';
$BOWTIE_INDEX = '/juno/depot/assemblies/S.cerevisiae/S288C_R64/index/bowtie/1.1.1/S288C_R64_bowtie';
$BOWTIE2_INDEX = '/juno/depot/assemblies/S.cerevisiae/S288C_R64/index/bowtie/2.2.4/S288C_R64_bowtie';
$chrSplits = '/juno/depot/assemblies/S.cerevisiae/S288C_R64/chromosomes';
$RIBOSOMAL_INTERVALS = "$Bin/data/S288C_R64/ribosomal_S288C_R64_assemblies.interval_file";
$REF_FLAT = "$Bin/data/S288C_R64/Saccharomyces_cerevisiae.R64-1-1.94.dedup.refflat.gz";
$TRANS_INDEX = '/juno/depot/assemblies/S.cerevisiae/S288C_R64/index/bowtie/2.2.4/transcriptome/ensembl/v94/Saccharomyces_cerevisiae.R64-1-1.94';
$TRANS_INDEX_DEDUP = '';
$TRANS_FASTA_DEDUP = '';
$geneNameConversion = "$Bin/data/S288C_R64/S288C_R64EnsemblIDToGeneName.txt";
$KALLISTO_INDEX = '/juno/depot/assemblies/S.cerevisiae/S288C_R64/index/kallisto/v0.42.1/ensembl/v94/Saccharomyces_cerevisiae.R64-1-1.94';
$RSEM_DB = '';
$STAR_FUSION_GENOME_LIB = '';
$CHIMERASCAN_FP_FILTER = '';
if($r1adaptor){
$starDB = '/juno/depot/assemblies/S.cerevisiae/S288C_R64/index/star/2.4.1d/ensembl/v94/overhang49';
}
else{
$starDB = '/juno/depot/assemblies/S.cerevisiae/S288C_R64/index/star/2.4.1d/ensembl/v94/overhang74';
}
$STAR_MAX_MEM = 30;
}
else{
die "Species $species is not currently supported in the rnaseq pipeline $!";
}
open(MA, "$map") or die "Can't open mapping file $map $!";
while(<MA>){
chomp;
my @data = split(/\s+/, $_);
if(!-d $data[3]){
die "$data[3] does not exist\n";
}
}
close MA;
my $htseq_stranded = '';
my $picard_strand_specificity = '';
my $star_outSAMstrandField = ''; ### compatibility with cufflinks;
my $cufflinks_lib_type = '';
if($strand =~ /none/i){
$htseq_stranded = 'no';
$picard_strand_specificity = 'NONE';
$star_outSAMstrandField = '--outSAMstrandField intronMotif';
$cufflinks_lib_type = '--library-type fr-unstranded';
}
elsif($strand =~ /forward/i){
$htseq_stranded = 'yes';
$picard_strand_specificity = 'FIRST_READ_TRANSCRIPTION_STRAND';
$cufflinks_lib_type = '--library-type fr-firststrand';
}
elsif($strand =~ /reverse/i){
$htseq_stranded = 'reverse';
$picard_strand_specificity = 'SECOND_READ_TRANSCRIPTION_STRAND';
$cufflinks_lib_type = '--library-type fr-secondstrand';
}
`/bin/mkdir -m 775 -p $output/intFiles`;
`/bin/mkdir -m 775 -p $output/progress`;
`/bin/mkdir -m 775 -p $output/gene`;
`/bin/mkdir -m 775 -p $output/gene/alignments`;
`/bin/mkdir -m 775 -p $output/metrics`;
`/bin/mkdir -m 775 -p $output/metrics/crm`;
`/bin/mkdir -m 775 -p $output/metrics/images`;
my %addParams = (scheduler => "$scheduler", runtime => "500", priority_project=> "$priority_project", priority_group=> "$priority_group", queues => "lau.q,lcg.q,nce.q", rerun => "1", iounits => "1");
my $additionalParams = Schedule::additionalParams(%addParams);
my @syncJobs = ();
open(IN, "$map") or die "Can't open $map $!";
while(<IN>){
chomp;
my @data = split(/\s+/, $_);
if(scalar(@included_samples) > 0){
my %incl = map { $_, 1 } @included_samples;
if( !$incl{ $data[1] } ){
print "EXCLUDING sample $data[1]\n";
next;
}
}
if($data[1] =~ /^\d+/){
$data[1] = "s_" . "$data[1]";
}
### sometimes in the mapping file,
### the lib, sample, and run id isn't a unique identifier
if($samp_libs_run{$data[1]}{$data[0]}{$data[2]}){
$slr_count++;
my @currentTime = &getTime();
print LOG "$currentTime[2]:$currentTime[1]:$currentTime[0], $currentTime[5]\/$currentTime[4]\/$currentTime[3]\tWARNING: $data[1]\t$data[0]\t$data[2] ISN'T UNIQUE; ";
$data[2] = "$data[2]\_$slr_count";
print LOG "WRITING INSTEAD TO $data[1]\/$data[0]\/$data[2]\n";
}
`/bin/mkdir -m 775 -p $output/intFiles/$data[1]`;
`/bin/mkdir -m 775 -p $output/intFiles/$data[1]/$data[0]/$data[2]`;
$samp_libs_run{$data[1]}{$data[0]}{$data[2]} = 1;
`ln -s $data[3]/* $output/intFiles/$data[1]/$data[0]/$data[2]/`;
chdir "$output/intFiles/$data[1]/$data[0]/$data[2]";
opendir(workDir, "./");
my @unsorted = readdir workDir;
closedir workDir;
my @files = sort @unsorted;
open(OUT, ">files_$data[1]\_$data[0]\_$data[2]");
foreach my $file (@files){
if($file =~ /fastq\.gz$/ && $file =~ m/^(.*)(R\d+)(.*)$/){
if($2 eq 'R1'){
my $file_R2 = $file;
$file_R2 =~ s/^(.*)R1(.*)$/$1R2$2/;
if($data[4] =~ /pe/i){
$samp_pair{$data[1]} = "PE";
print OUT "$file\t$file_R2\n";
}
elsif($data[4] =~ /se/i){
$samp_pair{$data[1]} = "SE";
print OUT "$file\n";
}
else{
die "CAN'T DETERMINE WHETHER RUN IS PAIRED OR SINGLE ENDED for sample $data[1]\n";
}
}
}
}
close OUT;
chdir $curDir;
}
close IN;
## QC sample key(s) and comparison file(s)
if($deseq || $clustering_only){
my $cluster_only = '';
if($clustering_only){
$cluster_only = '--clustering_only TRUE';
}
my $qc = `$R/Rscript $Bin/RunDE_inputQC.R --Rlibs $Rlibs --bin $Bin --pre $pre $cluster_only 2>&1`;
my $ec = $? >> 8;
print("$qc");
if($ec == 1){
die("Error(s) found in DESeq input files.");
}
}
my @crm = ();
my @crm_tophat = ();
my @asm = ();
my @asm_tophat = ();
my @ism = ();
my @ism_tophat = ();
my $ran_tophat = 0;
my @cag_jids = ();
my $ran_cag = 0;
my @thro_jids = ();
my $ran_tophatcrm = 0;
my @tophatcrm_jids = ();
my $ran_tophatasm = 0;
my $ran_tophatism = 0;
my $ran_tophatism_merge = 0;
my @tophatasm_jids = ();
my @tophatism_jids = ();
my $ran_rseqc = 0;
my $ran_rseqc_merge = 0;
my $ran_plots = 0;
my @rseqc_jids = ();
my $ran_picard_metrics = 0;
my $ran_starcrm = 0;
my @starcrm_jids = ();
my $ran_starasm = 0;
my @starasm_jids = ();
my $ran_starism = 0;
my $ran_starism_merge = 0;
my @starism_jids = ();
my $ran_star = 0;
my @staraddrg_jids = ();
my $ran_tophathtseq = 0;
my @tophathtseq_jids = ();
my $ran_tophatdexseq = 0;
my @tophatdexseq_jids = ();
my $ran_starhtseq = 0;
my @starhtseq_jids = ();
my $ran_stardexseq = 0;
my @stardexseq_jids = ();
my $ran_rceg = 0;
my @rce_jids = ();
my $ran_kallisto = 0;
my @kallisto_jids = ();
my @qcplot_jids = ();
my @qcpdf_jids = ();
foreach my $sample (keys %samp_libs_run){
my @R1 = ();
my @R2 = ();
my $readsFlag = 0;
my $grl = 0;
my $minReadLength = -1;
my $sampReadLength = 0;
foreach my $lib (keys %{$samp_libs_run{$sample}}){
foreach my $run (keys %{$samp_libs_run{$sample}{$lib}}){
open(READS, "$output/intFiles/$sample/$lib/$run/files_$sample\_$lib\_$run") || die "Can't open $output/intFiles/$sample/$lib/$run/files_$sample\_$lib\_$run $!";
while(<READS>){
chomp;
my @readPair = split(/\s+/, $_);
### NOTE: JUST CHECKING TO SEE IF IT EXISTS
### HOWEVER DOES NOT GURANTEE THAT IT'S NON-EMPTY
if(-e "$output/intFiles/$sample/$lib/$run/$readPair[0]"){
push @R1, "$output/intFiles/$sample/$lib/$run/$readPair[0]";
if($samp_pair{$sample} eq "PE"){
if(!-e "$output/intFiles/$sample/$lib/$run/$readPair[1]"){
$readsFlag = 1;
}
push @R2, "$output/intFiles/$sample/$lib/$run/$readPair[1]";
}
if($grl == 0){
my $readO = `/bin/gzip -cd $output/intFiles/$sample/$lib/$run/$readPair[0] | head -2`;
### discard reads less than half of original read length by cutadapt
#my $readO = `/usr/bin/head -2 $output/intFiles/$readPair[0]`;
chomp $readO;
my @dataO = split(/\n/, $readO);
my $readLength = length($dataO[1]);
if($sampReadLength > 0 & $readLength != $sampReadLength){
print "WARNING: fastqs have variable read lengths\n";
}
$sampReadLength = $readLength;
$minReadLength = int(0.5*$readLength);
$grl = 1;
}
}
else{
$readsFlag = 1;
}
}
close READS;
}
}
if($readsFlag == 1){
my @currentTime = &getTime();
print LOG "$currentTime[2]:$currentTime[1]:$currentTime[0], $currentTime[5]\/$currentTime[4]\/$currentTime[3]\tSKIPPING SAMPLE $sample ANALYSIS BECAUSE CAN'T LOCATE ALL LISTED READS";
next;
}
my $r1_gz_files = join(",", @R1);
my $r2_gz_files = join(",", @R2);
my @gz_jids = ();