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neoscan.v1.1.pl
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#########Song Cao###########
# last updated date: 12/21/2016 #
### updated 1/3/2018 ###
### updated 08/11/2018 ##
### use docker image for submitting jobs to research-hpc queue ##
#!/usr/bin/perl
use strict;
use warnings;
#use POSIX;
my $version = 1.0;
#color code
my $red = "\e[31m";
my $gray = "\e[37m";
my $yellow = "\e[33m";
my $green = "\e[32m";
my $purple = "\e[35m";
my $cyan = "\e[36m";
my $normal = "\e[0m";
#usage information
(my $usage = <<OUT) =~ s/\t+//g;
This script will predict neoantigen for somatic variants in cancer sample
Pipeline version: $version
$yellow Usage: perl $0 <run_folder> <step_number> $normal
<run_folder> = full path of the folder holding files for this sequence run
<step_number> run this pipeline step by step. (running the whole pipeline if step number is 0)
$red [1] Generate fasta snv and indel
[2] Generate peptide for snv and indel
$green [3] Run BWA
[4] parse bwa
$purple [5] Run netMHC
[6] parse netMHC result
$yellow [7] get ref read count
[8] Generate final report
$normal
OUT
die $usage unless @ARGV == 2;
my ( $run_dir, $step_number ) = @ARGV;
if ($run_dir =~/(.+)\/$/) {
$run_dir = $1;
}
die $usage unless ($step_number >=0)&&(($step_number <= 10) || ($step_number>=12));
my $email = "scao\@wustl\.edu";
my $HOME = $ENV{HOME};
my $working_name= (split(/\//,$run_dir))[-1];
my $HOME1="/gscmnt/gc2524/dinglab";
#store job files here
if (! -d $HOME1."/tmpNeo") {
`mkdir $HOME1"/tmpNeo"`;
}
my $job_files_dir = $HOME1."/tmpNeo";
#store SGE output and error files here
if (! -d $HOME1."/LSF_DIR_Neo") {
`mkdir $HOME1"/LSF_DIR_Neo"`;
}
my $lsf_file_dir = $HOME1."/LSF_DIR_Neo";
my $db_hla_abc_cds="/gscmnt/gc2523/dinglab/neoantigen/human_DB/HLA_ABC_CDS.fasta";
my $hlaminer="/gscmnt/gc2523/dinglab/neoantigen/HLAminer_v1.3.1/bin/HLAminer_song.pl";
my $hla_nom_p="/gscmnt/gc2523/dinglab/neoantigen/HLAminer_v1.3.1/database/hla_nom_p.txt";
my $f_allele="/gscmnt/gc2523/dinglab/neoantigen/netMHC-4.0/Linux_x86_64/data/allelelist";
my $netMHC="/gscmnt/gc2523/dinglab/neoantigen/netMHC-4.0/netMHC";
my $db_ref_bed="/gscmnt/gc2524/dinglab/PepScan_Runs/BRCA/proteogenomics/databases/refseq_human_20130727_proteome/proteome.bed";
my $h37_fa="/gscmnt/gc2524/dinglab/PepScan_Runs/BRCA/proteogenomics/databases/genome_human";
my $db_cdna="/gscmnt/gc3027/dinglab/medseq/fasta/human/Homo_sapiens.GRCh37.70.cdna.all.fa";
my $db_sanger_qs="/gscmnt/gc2523/dinglab/neoantigen/neoantigen-scan/quality_sanger.table.2col.tsv";
#my $f_h37="/gscmnt/gc2522/dinglab/ref_tophat/bowtie2-index/Homo_sapiens.GRCh37.75";
#my $f_h37_gtf="/gscmnt/gc2522/dinglab/ref_tophat/bowtie2-index/Homo_sapiens.GRCh37.75.gtf";
#my $f_g="/gscmnt/gc2522/dinglab/ref_tophat/Bowtie2Index/genome";
# obtain script path
my $run_script_path = `dirname $0`;
chomp $run_script_path;
my $run_script_path_perl = "/usr/bin/perl ".$run_script_path."/";
my $run_script_path_python = "/usr/bin/python ".$run_script_path."/";
#my $hold_RM_job = "norm";
my $current_job_file = "";#cannot be empty
my $hold_job_file = "";
my $bsub_com = "";
my $sample_full_path = "";
my $sample_name = "";
opendir(DH, $run_dir) or die "Cannot open dir $run_dir: $!\n";
my @sample_dir_list = readdir DH;
close DH;
if ($step_number < 8 || $step_number>=12) {
#begin to process each sample
for (my $i=0;$i<@sample_dir_list;$i++) {#use the for loop instead. the foreach loop has some problem to pass the global variable $sample_name to the sub functions
$sample_name = $sample_dir_list[$i];
if (!($sample_name =~ /\./ || $sample_name=~/worklog/)) {
$sample_full_path = $run_dir."/".$sample_name;
if (-d $sample_full_path) { # is a full path directory containing a sample
print $yellow, "\nSubmitting jobs for the sample ",$sample_name, "...",$normal, "\n";
$current_job_file="";
if($step_number == 0 || $step_number>=12)
{
if($step_number==0)
{
&bsub_fa();}
if($step_number<=12)
{
&bsub_pep(); }
if($step_number<=13)
{
&bsub_bwa();}
if($step_number<=14)
{
&bsub_parse_bwa();}
if($step_number<=15)
{
&bsub_netmhc();}
if($step_number<=16)
{
&bsub_parsemhc();}
if($step_number<=17)
{
&bsub_get_ref_count();}
}
elsif ($step_number == 1) {
&bsub_fa();
}
elsif ($step_number == 2) {
&bsub_pep(1);
} elsif ($step_number == 3) {
&bsub_bwa(1);
} elsif ($step_number == 4) {
&bsub_parse_bwa(1);
} elsif ($step_number == 5) {
&bsub_netmhc(1);
}elsif ($step_number == 6) {
&bsub_parsemhc(1);
} elsif ($step_number == 7) {
&bsub_get_ref_count(1);
}
}
}
}
}
if($step_number==0 || $step_number>=12)
{
print "generate the final report\n";
&bsub_final_report();
}
if($step_number==8)
{
print "generate the final report\n";
&bsub_final_report(1);
}
#######################################################################
# send email to notify the finish of the analysis
if (($step_number == 0) || ($step_number == 9)) {
print $yellow, "Submitting the job for sending an email when the run finishes ",$sample_name, "...",$normal, "\n";
$hold_job_file = $current_job_file;
$current_job_file = "Email_run_".$$.".sh";
open(EMAIL, ">$job_files_dir/$current_job_file") or die $!;
print EMAIL "#!/bin/bash\n";
print EMAIL "#BSUB -n 1\n";
print EMAIL "#BSUB -o $lsf_file_dir","\n";
print EMAIL "#BSUB -e $lsf_file_dir","\n";
print EMAIL "#BSUB -J $current_job_file\n";
print EMAIL "#BSUB -w \"$hold_job_file\"","\n";
print EMAIL "#BSUB -q ding-lab\n";
print EMAIL $run_script_path."send_email.pl ".$run_dir." ".$email."\n";
close EMAIL;
$bsub_com = "bsub < $job_files_dir/$current_job_file\n";
#$bsub_com = "qsub -V -hold_jid $hold_job_file -e $lsf_file_dir -o $lsf_file_dir $job_files_dir/$current_job_file\n";
system ($bsub_com);
}
#######################################################################
if ($step_number == 0) {
print $green, "All jobs are submitted! You will get email notification when this run is completed.\n",$normal;
}
### step 1: get fasta sequence for transcript with indel and snv mutation ##
sub bsub_fa{
#my $cdhitReport = $sample_full_path."/".$sample_name.".fa.cdhitReport";
$current_job_file = "j1_fa_".$sample_name.".sh";
#my $IN_sam = $sample_full_path."/".$sample_name.".exome.sam";
my $lsf_out=$lsf_file_dir."/".$current_job_file.".out";
my $lsf_err=$lsf_file_dir."/".$current_job_file.".err";
`rm $lsf_out`;
`rm $lsf_err`;
open(FA, ">$job_files_dir/$current_job_file") or die $!;
print FA "#!/bin/bash\n";
#print FA "#BSUB -n 1\n";
#print FA "#BSUB -R \"rusage[mem=30000]\"","\n";
#print FA "#BSUB -M 30000000\n";
# print FA "#BSUB -o $lsf_file_dir","/","$current_job_file.out\n";
# print FA "#BSUB -e $lsf_file_dir","/","$current_job_file.err\n";
# print FA "#BSUB -q ding-lab\n";
print FA "#BSUB -J $current_job_file\n";
# print FA "#BSUB -w \"$hold_job_file\"","\n";
print FA "f_vcf_indel=".$sample_full_path."/$sample_name.indel.vcf\n";
print FA "f_vcf_snp=".$sample_full_path."/$sample_name.snp.vcf\n";
print FA "f_fa_indel_wt=".$sample_full_path."/$sample_name.indel.vcf.fa-indel-wt.fasta\n";
print FA "f_fa_indel_mut=".$sample_full_path."/$sample_name.indel.vcf.fa-indel-mut.fasta\n";
print FA "f_fa_snp_wt=".$sample_full_path."/$sample_name.snp.vcf.fa-snv-wt.fasta\n";
print FA "f_fa_snp_mut=".$sample_full_path."/$sample_name.snp.vcf.fa-snv-mut.fasta\n";
print FA "f_fa_all=".$sample_full_path."/$sample_name.transcript.fa\n";
print FA 'if [ -s $f_vcf_snp ]',"\n";
print FA "then\n";
print FA " ".$run_script_path_perl."fasta_seq_for_snv_using_refseq_bed.pl $db_ref_bed $h37_fa \${f_vcf_snp}"."\n";
print FA "cat \${f_fa_snp_mut} > \${f_fa_all}","\n";
print FA "fi\n\n";
print FA 'if [ -s $f_vcf_indel ]',"\n";
print FA "then\n";
print FA " ".$run_script_path_perl."fasta_seq_for_indel_using_refseq_bed.pl $db_ref_bed $h37_fa \${f_vcf_indel}"."\n";
print FA "cat \${f_fa_indel_mut} >> \${f_fa_all}","\n";
print FA "fi\n\n";
print FA "";
close FA;
#$bsub_com = "bsub < $job_files_dir/$current_job_file\n";
my $sh_file=$job_files_dir."/".$current_job_file;
$bsub_com = "bsub -q research-hpc -n 1 -R \"select[mem>30000] rusage[mem=30000]\" -M 30000000 -a \'docker(registry.gsc.wustl.edu/genome/genome_perl_environment)\' -o $lsf_out -e $lsf_err sh $sh_file\n";
system ( $bsub_com );
}
##step 2: get peptide sequences for transcript with indel and snv mutations ##
sub bsub_pep{
my ($step_by_step) = @_;
if ($step_by_step) {
$hold_job_file = "";
}
else{
$hold_job_file = $current_job_file;
}
#my $cdhitReport = $sample_full_path."/".$sample_name.".fa.cdhitReport";
$current_job_file = "j2_pep_".$sample_name.".sh";
my $lsf_out=$lsf_file_dir."/".$current_job_file.".out";
my $lsf_err=$lsf_file_dir."/".$current_job_file.".err";
`rm $lsf_out`;
`rm $lsf_err`;
#my $IN_sam = $sample_full_path."/".$sample_name.".exome.sam";
open(PEP, ">$job_files_dir/$current_job_file") or die $!;
print PEP "#!/bin/bash\n";
# print PEP "#BSUB -n 1\n";
# print PEP "#BSUB -R \"rusage[mem=30000]\"","\n";
# print PEP "#BSUB -M 30000000\n";
# print PEP "#BSUB -q ding-lab\n";
# print PEP "#BSUB -o $lsf_file_dir","/","$current_job_file.out\n";
# print PEP "#BSUB -e $lsf_file_dir","/","$current_job_file.err\n";
# print PEP "#BSUB -J $current_job_file\n";
# print PEP "#BSUB -w \"$hold_job_file\"","\n";
print PEP "f_vcf_indel=".$sample_full_path."/$sample_name.indel.vcf\n";
print PEP "f_vcf_snp=".$sample_full_path."/$sample_name.snp.vcf\n";
print PEP "f_pep_indel_wt=".$sample_full_path."/$sample_name.indel.vcf.proteome-indel-wt.fasta\n";
print PEP "f_pep_indel_mut=".$sample_full_path."/$sample_name.indel.vcf.proteome-indel-mut.fasta\n";
print PEP "f_pep_snp_wt=".$sample_full_path."/$sample_name.snp.vcf.proteome-snv-wt.fasta\n";
print PEP "f_pep_snp_mut=".$sample_full_path."/$sample_name.snp.vcf.proteome-snv-mut.fasta\n";
print PEP "f_pep_all=".$sample_full_path."/$sample_name.pep.fa\n";
print PEP 'if [ -s $f_vcf_snp ]',"\n";
print PEP "then\n";
print PEP " ".$run_script_path_perl."protein_seq_for_snv_using_refseq_bed.pl $db_ref_bed $h37_fa \${f_vcf_snp}"."\n";
print PEP "cat \${f_pep_snp_mut} > \${f_pep_all}","\n";
print PEP "fi\n\n";
print PEP 'if [ -s $f_vcf_indel ]',"\n";
print PEP "then\n";
print PEP " ".$run_script_path_perl."protein_seq_for_indel_using_refseq_bed.pl $db_ref_bed $h37_fa \${f_vcf_indel}"."\n";
print PEP "cat \${f_pep_indel_mut} >> \${f_pep_all}","\n";
print PEP "fi\n\n";
print PEP "";
close PEP;
my $sh_file=$job_files_dir."/".$current_job_file;
# $bsub_com = "bsub -q research-hpc -n 1 -R \"select[mem>30000] rusage[mem=30000]\" -M 30000000 -a \'docker(registry.gsc.wustl.edu/genome/genome_perl_environment)\' -w $hold_job_file -o $lsf_out -e $lsf_err sh $sh_file\n";
$bsub_com = "bsub -q research-hpc -n 1 -R \"select[mem>30000] rusage[mem=30000]\" -M 30000000 -a \'docker(registry.gsc.wustl.edu/genome/genome_perl_environment)\' -o $lsf_out -e $lsf_err sh $sh_file\n";
#$bsub_com = "bsub < $job_files_dir/$current_job_file\n";
system ( $bsub_com );
}
##step 3: use bwa to align the hla reference to get HLA genotypet ##
sub bsub_bwa{
#my $cdhitReport = $sample_full_path."/".$sample_name.".fa.cdhitReport";
my ($step_by_step) = @_;
if ($step_by_step) {
$hold_job_file = "";
}else{
$hold_job_file = $current_job_file;
}
$current_job_file = "j3_bwa_".$sample_name.".sh";
my $lsf_out=$lsf_file_dir."/".$current_job_file.".out";
my $lsf_err=$lsf_file_dir."/".$current_job_file.".err";
`rm $lsf_out`;
`rm $lsf_err`;
my $IN_bam = $sample_full_path."/".$sample_name.".rnaseq.bam";
my $f_fq_1=$sample_full_path."/".$sample_name.".1.fq";
my $f_fq_2=$sample_full_path."/".$sample_name.".2.fq";
# my $RNASEQ_MT_sam=$sample_full_path."/".$sample_name.".rnaseq.mapped.sam";
if ((! -e $IN_bam) && ((!-e $f_fq_1) || (! -e $f_fq_2))) {#make sure there is a input fasta file
print $red, "&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&\n";
print "Warning: Died because there is no input bam or fq file for bwa:\n";
print "File $IN_bam does not exist!\n";
die "Please check command line argument!", $normal, "\n\n";
}
#if (! -s $IN_bam) {#make sure input fasta file is not empty
# print $red, "&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&\n";
# die "Warning: Died because $IN_bam is empty!", $normal, "\n\n";
#}
open(RNASEQ, ">$job_files_dir/$current_job_file") or die $!;
# my $f_fq_1=$sample_full_path."/".$sample_name.".1.fq";
# my $f_fq_2=$sample_full_path."/".$sample_name.".2.fq";
print RNASEQ "#!/bin/bash\n";
#print RNASEQ "#BSUB -n 1\n";
#print RNASEQ "#BSUB -R \"rusage[mem=30000]\"","\n";
#print RNASEQ "#BSUB -M 30000000\n";
#print RNASEQ "#BSUB -q ding-lab\n";
#print RNASEQ "#BSUB -w \"$hold_job_file\"","\n";
#print RNASEQ "#BSUB -o $lsf_file_dir","/","$current_job_file.out\n";
#print RNASEQ "#BSUB -e $lsf_file_dir","/","$current_job_file.err\n";
#print RNASEQ "#BSUB -J $current_job_file\n";
print RNASEQ "RNASEQ_IN=".$sample_full_path."/".$sample_name.".rnaseq.bam\n";
print RNASEQ "RNASEQ_sorted=".$sample_full_path."/".$sample_name.".rnaseq.sorted\n";
print RNASEQ "RNASEQ_sorted_bam=".$sample_full_path."/".$sample_name.".rnaseq.sorted.bam\n";
print RNASEQ "UNMAPPED_IN=".$sample_full_path."/".$sample_name.".rnaseq.unaligned_reads.bam\n";
print RNASEQ "UNMAPPED_sorted=".$sample_full_path."/".$sample_name.".rnaseq.unaligned_reads.sorted\n";
print RNASEQ "UNMAPPED_sorted_bam=".$sample_full_path."/".$sample_name.".rnaseq.unaligned_reads.sorted.bam\n";
print RNASEQ "RNASEQ_fq_1=".$sample_full_path."/".$sample_name.".1.fq\n";
print RNASEQ "RNASEQ_fq_2=".$sample_full_path."/".$sample_name.".2.fq\n";
print RNASEQ "RNASEQ_sai_1=".$sample_full_path."/".$sample_name.".1.sai\n";
print RNASEQ "RNASEQ_sai_2=".$sample_full_path."/".$sample_name.".2.sai\n";
print RNASEQ "RNASEQ_sam=".$sample_full_path."/".$sample_name.".rnaseq.sam\n";
print RNASEQ "INDEX_T=".$sample_full_path."/".$sample_name.".transcript.fa\n";
print RNASEQ "RNASEQ_sai_T1=".$sample_full_path."/".$sample_name.".T1.sai\n";
print RNASEQ "RNASEQ_sai_T2=".$sample_full_path."/".$sample_name.".T2.sai\n";
print RNASEQ "RNASEQ_T_sam=".$sample_full_path."/".$sample_name.".T.rnaseq.sam\n";
print RNASEQ "HLA_log=".$sample_full_path."/HLAminer_HPRA.log\n";
print RNASEQ "HLA_csv=".$sample_full_path."/HLAminer_HPRA.csv\n";
print RNASEQ "HLA_tsv=".$sample_full_path."/HLAminer_alleles.tsv\n";
#print HLA "HLA_bam=".$sample_full_path."/$sample_name.hla.bam\n";
##mapped reads##
print RNASEQ 'if [ -f $IN_bam ]',"\n"; # input file exist
print RNASEQ "then\n";
print RNASEQ "samtools sort -n \${RNASEQ_IN} \${RNASEQ_sorted}","\n";
print RNASEQ "samtools view \${RNASEQ_sorted_bam} | perl -ne \'\$l=\$_; \$f_q1=\"$f_fq_1\"; \$f_q2=\"$f_fq_2\"; if(\$first==0) { open(OUT1,\">\$f_q1\"); open(OUT2,\">\$f_q2\"); \$first=1;} \@ss=split(\"\\t\",\$l); \$flag=\$ss[1]; \$cigar=\$ss[5]; if((\$flag & 0x100) || (\$flag & 0x800) || (\$cigar=~/H/)) { next; } \$id=\$ss[0]; \$seq=\$ss[9]; \$q=\$ss[10]; if(\$id=~/\\/1\$/ || (\$flag & 0x40) ) { \$r1=\$id; \$r1=~s/\\/1\$//g; \$seq1=\$seq; \$q1=\$q; } if(\$id=~/\\/2\$/ || (\$flag & 0x80)) { \$r2=\$id; \$r2=~s/\\/2\$//g; \$seq2=\$seq; \$q2=\$q; } if((\$r1 eq \$r2)) { print OUT1 \"\@\",\$r1,\"/1\",\"\\n\"; print OUT1 \$seq1,\"\\n\"; print OUT1 \"+\",\"\\n\"; print OUT1 \$q1,\"\\n\"; print OUT2 \"\@\",\$r1,\"/2\",\"\\n\"; print OUT2 \$seq2,\"\\n\"; print OUT2 \"+\",\"\\n\"; print OUT2 \$q2,\"\\n\";}\'","\n";
print RNASEQ " fi\n\n";
### unmapped reads ###
print RNASEQ 'if [ -f $UNMAPPED_IN ]',"\n"; # input file exist
print RNASEQ "then\n";
print RNASEQ "samtools sort -n \${UNMAPPED_IN} \${UNMAPPED_sorted}","\n";
print RNASEQ "samtools view \${UNMAPPED_sorted_bam} | perl -ne \'\$l=\$_; \$f_q1=\"$f_fq_1\"; \$f_q2=\"$f_fq_2\"; if(\$first2==0) { open(OUT1,\">>\$f_q1\"); open(OUT2,\">>\$f_q2\"); \$first2=1;} \@ss=split(\"\\t\",\$l); \$flag=\$ss[1]; \$cigar=\$ss[5]; if((\$flag & 0x100) || (\$flag & 0x800) || (\$cigar=~/H/)) { next; } \$id=\$ss[0]; \$seq=\$ss[9]; \$q=\$ss[10]; if(\$id=~/\\/1\$/ || (\$flag & 0x40) ) { \$r1=\$id; \$r1=~s/\\/1\$//g; \$seq1=\$seq; \$q1=\$q; } if(\$id=~/\\/2\$/ || (\$flag & 0x80)) { \$r2=\$id; \$r2=~s/\\/2\$//g; \$seq2=\$seq; \$q2=\$q; } if((\$r1 eq \$r2)) { print OUT1 \"\@\",\$r1,\"/1\",\"\\n\"; print OUT1 \$seq1,\"\\n\"; print OUT1 \"+\",\"\\n\"; print OUT1 \$q1,\"\\n\"; print OUT2 \"\@\",\$r1,\"/2\",\"\\n\"; print OUT2 \$seq2,\"\\n\"; print OUT2 \"+\",\"\\n\"; print OUT2 \$q2,\"\\n\";}\'","\n";
#print RNASEQ "bwa0.7.12 mem -t 8 $db_hla_abc_cds \${RNASEQ_fq_1} \${RNASEQ_fq_2} > \${RNASEQ_sam}"."\n";
print RNASEQ " fi\n\n";
### bwa alignement ###
#print RNASEQ "bwa0.7.12 index \${INDEX_T}"."\n";
print RNASEQ "rm \${RNASEQ_sorted_bam}","\n";
print RNASEQ 'if [ -f $UNMAPPED_sorted_bam ]',"\n"; # input file exist
print RNASEQ "then\n";
print RNASEQ "rm \${UNMAPPED_sorted_bam}","\n";
print RNASEQ "fi\n";
print RNASEQ "bwa0.7.12 aln -e 0 -o 0 -t 4 -f \${RNASEQ_sai_1} $db_hla_abc_cds \${RNASEQ_fq_1}"."\n";
print RNASEQ "bwa0.7.12 aln -e 0 -o 0 -t 4 -f \${RNASEQ_sai_2} $db_hla_abc_cds \${RNASEQ_fq_2}"."\n";
print RNASEQ "bwa0.7.12 sampe -f \${RNASEQ_sam} $db_hla_abc_cds \${RNASEQ_sai_1} \${RNASEQ_sai_2} \${RNASEQ_fq_1} \${RNASEQ_fq_2}"."\n";
print RNASEQ "perl $hlaminer -h $db_hla_abc_cds -p $hla_nom_p -a \${RNASEQ_sam} -o $sample_full_path"."\n";
print RNASEQ " ".$run_script_path_perl."parseHLAresult.pl \${HLA_csv} \${HLA_tsv}"."\n";
print RNASEQ "rm \${RNASEQ_sam}","\n";
print RNASEQ "bwa0.7.12 index \${INDEX_T}"."\n";
print RNASEQ "bwa0.7.12 aln -e 0 -o 0 -t 4 -f \${RNASEQ_sai_T1} \${INDEX_T} \${RNASEQ_fq_1}"."\n";
print RNASEQ "bwa0.7.12 aln -e 0 -o 0 -t 4 -f \${RNASEQ_sai_T2} \${INDEX_T} \${RNASEQ_fq_2}"."\n";
print RNASEQ "bwa0.7.12 sampe -f \${RNASEQ_T_sam} \${INDEX_T} \${RNASEQ_sai_T1} \${RNASEQ_sai_T2} \${RNASEQ_fq_1} \${RNASEQ_fq_2}"."\n";
print RNASEQ "rm \${RNASEQ_fq_1}","\n";
print RNASEQ "rm \${RNASEQ_fq_2}","\n";
print RNASEQ "rm \${RNASEQ_sai_1}","\n";
print RNASEQ "rm \${RNASEQ_sai_2}","\n";
print RNASEQ "rm \${RNASEQ_sai_T1}","\n";
print RNASEQ "rm \${RNASEQ_sai_T2}","\n";
close RNASEQ;
my $sh_file=$job_files_dir."/".$current_job_file;
# $bsub_com = "bsub -q research-hpc -n 1 -R \"select[mem>30000] rusage[mem=30000]\" -M 30000000 -a \'docker(registry.gsc.wustl.edu/genome/genome_perl_environment)\' -w $hold_job_file -o $lsf_out -e $lsf_err sh $sh_file\n";
$bsub_com = "bsub -q research-hpc -n 1 -R \"select[mem>30000] rusage[mem=30000]\" -M 30000000 -a \'docker(registry.gsc.wustl.edu/genome/genome_perl_environment)\' -w \"$hold_job_file\" -o $lsf_out -e $lsf_err sh $sh_file\n";
#$bsub_com = "bsub < $job_files_dir/$current_job_file\n";
system ( $bsub_com );
#$bsub_com = "bsub < $job_files_dir/$current_job_file\n";
#system ( $bsub_com );
}
sub bsub_parse_bwa{
#my $cdhitReport = $sample_full_path."/".$sample_name.".fa.cdhitReport";
my ($step_by_step) = @_;
if ($step_by_step) {
$hold_job_file = "";
}else{
$hold_job_file = $current_job_file;
}
#my $RNASEQ_MT_sam=$sample_full_path."/".$sample_name.".rnaseq.mapped.sam";
$current_job_file = "j4_parse_bwa_".$sample_name.".sh";
my $lsf_out=$lsf_file_dir."/".$current_job_file.".out";
my $lsf_err=$lsf_file_dir."/".$current_job_file.".err";
`rm $lsf_out`;
`rm $lsf_err`;
my $RNASEQ_MT_sam=$sample_full_path."/".$sample_name.".rnaseq.T.mapped.sam";
open(BWAP, ">$job_files_dir/$current_job_file") or die $!;
print BWAP "#!/bin/bash\n";
#print BWAP "#BSUB -n 1\n";
#print BWAP "#BSUB -R \"rusage[mem=30000]\"","\n";
#print BWAP "#BSUB -q ding-lab\n";
#print BWAP "#BSUB -M 30000000\n";
#print BWAP "#BSUB -o $lsf_file_dir","/","$current_job_file.out\n";
#print BWAP "#BSUB -e $lsf_file_dir","/","$current_job_file.err\n";
#print BWAP "#BSUB -J $current_job_file\n";
#print BWAP "#BSUB -w \"$hold_job_file\"","\n";
print BWAP "RNASEQ_T_sam=".$sample_full_path."/".$sample_name.".T.rnaseq.sam\n";
print BWAP "CDNA_sai=".$sample_full_path."/".$sample_name.".cdna.sai\n";
print BWAP "CDNA_sam=".$sample_full_path."/".$sample_name.".cdna.sam\n";
print BWAP "RNASEQ_T_fq=".$sample_full_path."/".$sample_name.".T.rnaseq.fq\n";
print BWAP "RNASEQ_TF_fq=".$sample_full_path."/".$sample_name.".T.rnaseq.filtered.fq\n";
print BWAP "f_fa_indel_wt=".$sample_full_path."/$sample_name.indel.vcf.fa-indel-wt.fasta\n";
print BWAP "f_fa_indel_mut=".$sample_full_path."/$sample_name.indel.vcf.fa-indel-mut.fasta\n";
print BWAP "f_fa_snp_wt=".$sample_full_path."/$sample_name.snp.vcf.fa-snv-wt.fasta\n";
print BWAP "f_fa_snp_mut=".$sample_full_path."/$sample_name.snp.vcf.fa-snv-mut.fasta\n";
print BWAP "f_novel_seq=".$sample_full_path."/$sample_name.novelseq.fa\n";
print BWAP "f_novel_q20_seq=".$sample_full_path."/$sample_name.novelseq.q20.fa\n";
print BWAP "f_indel_pos=".$sample_full_path."/$sample_name.indel.fa.pos.tsv\n";
print BWAP "f_snv_pos=".$sample_full_path."/$sample_name.snv.fa.pos.tsv\n";
print BWAP "f_mut_pos=".$sample_full_path."/$sample_name.mut.fa.pos.tsv\n";
print BWAP "f_fa_all=".$sample_full_path."/$sample_name.transcript.fa\n";
print BWAP "f_sup=".$sample_full_path."/$sample_name.transcript.novel.support.rna.tsv\n";
print BWAP " ".$run_script_path_perl."get_perferct_mapped_reads.pl \${RNASEQ_T_sam} \${RNASEQ_T_fq}"."\n";
print BWAP "bwa0.7.12 aln $db_cdna \${RNASEQ_T_fq} > \${CDNA_sai}"."\n";
print BWAP "bwa0.7.12 samse $db_cdna \${CDNA_sai} \${RNASEQ_T_fq} > \${CDNA_sam}"."\n";
print BWAP " ".$run_script_path_perl."filter_reads_in_cdna.pl \${CDNA_sam} \${RNASEQ_T_fq} \${RNASEQ_TF_fq}"."\n";
print BWAP " ".$run_script_path_perl."extract_novel_sequence.pl \${f_fa_all} \${f_fa_snp_wt} \${f_fa_indel_wt} \${RNASEQ_TF_fq} \${f_novel_seq}"."\n";
print BWAP " ".$run_script_path_perl."q20_filter.pl $db_sanger_qs \${f_novel_seq} \${RNASEQ_TF_fq} \${f_novel_q20_seq}"."\n";
print BWAP 'if [ -s $f_fa_indel_wt ]',"\n";
print BWAP "then\n";
print BWAP " ".$run_script_path_perl."find_first_diff_pos_mut_wt_indel.pl \${f_fa_indel_wt} \${f_fa_indel_mut} \${f_indel_pos}"."\n";
print BWAP "fi\n\n";
print BWAP " ".$run_script_path_perl."find_first_diff_pos_mut_wt_snv.pl \${f_fa_snp_wt} \${f_fa_snp_mut} \${f_snv_pos}"."\n";
print BWAP "cat \${f_indel_pos} \${f_snv_pos} > \${f_mut_pos}","\n";
print BWAP " ".$run_script_path_perl."extract_support_reads_from_novel_sequence.pl \${f_fa_all} \${f_novel_q20_seq} \${f_mut_pos} \${f_sup}"."\n";
print BWAP "rm \${RNASEQ_T_sam}","\n";
print BWAP "rm \${CDNA_sam}","\n";
close BWAP;
my $sh_file=$job_files_dir."/".$current_job_file;
# $bsub_com = "bsub -q research-hpc -n 1 -R \"select[mem>30000] rusage[mem=30000]\" -M 30000000 -a \'docker(registry.gsc.wustl.edu/genome/genome_perl_environment)\' -w $hold_job_file -o $lsf_out -e $lsf_err sh $sh_file\n";
$bsub_com = "bsub -q research-hpc -n 1 -R \"select[mem>30000] rusage[mem=30000]\" -M 30000000 -a \'docker(registry.gsc.wustl.edu/genome/genome_perl_environment)\' -w \"$hold_job_file\" -o $lsf_out -e $lsf_err sh $sh_file\n";
#$bsub_com = "bsub < $job_files_dir/$current_job_file\n";
system ( $bsub_com );
#$bsub_com = "bsub < $job_files_dir/$current_job_file\n";
#system ( $bsub_com );
}
sub bsub_netmhc{
my ($step_by_step) = @_;
if ($step_by_step) {
$hold_job_file = "";
}else{
$hold_job_file = $current_job_file;
}
#my $cdhitReport = $sample_full_path."/".$sample_name.".fa.cdhitReport";
$current_job_file = "j5_bind_".$sample_name.".sh";
my $lsf_out=$lsf_file_dir."/".$current_job_file.".out";
my $lsf_err=$lsf_file_dir."/".$current_job_file.".err";
`rm $lsf_out`;
`rm $lsf_err`;
#my $IN_sam = $sample_full_path."/".$sample_name.".exome.sam";
open(MHC, ">$job_files_dir/$current_job_file") or die $!;
print MHC "#!/bin/bash\n";
#print MHC "#BSUB -n 1\n";
#print MHC "#BSUB -R \"rusage[mem=30000]\"","\n";
#print MHC "#BSUB -M 30000000\n";
#print MHC "#BSUB -q ding-lab\n";
# print MHC "#BSUB -o $lsf_file_dir","/","$current_job_file.out\n";
# print MHC "#BSUB -e $lsf_file_dir","/","$current_job_file.err\n";
# print MHC "#BSUB -J $current_job_file\n";
# print MHC "#BSUB -w \"$hold_job_file\"","\n";
print MHC "f_pep=".$sample_full_path."/$sample_name.pep.fa\n";
print MHC "HLA_tsv=".$sample_full_path."/HLAminer_alleles.tsv\n";
print MHC "f_netMHC_result=".$sample_full_path."/netMHC4.0.out.append.txt\n";
print MHC "f_out=".$sample_full_path."/result_neoantigen\n";
print MHC " ".$run_script_path_python."runNetMHC4.py -a \${HLA_tsv} -f \${f_pep} -p 8,9,10,11 -o $sample_full_path -n $netMHC -v $f_allele"."\n";
close MHC;
my $sh_file=$job_files_dir."/".$current_job_file;
# $bsub_com = "bsub -q research-hpc -n 1 -R \"select[mem>30000] rusage[mem=30000]\" -M 30000000 -a \'docker(registry.gsc.wustl.edu/genome/genome_perl_environment)\' -o $lsf_out -e $lsf_err sh $sh_file\n";
$bsub_com = "LSF_DOCKER_PRESERVE_ENVIRONMENT=false bsub -q research-hpc -n 1 -R \"select[mem>30000] rusage[mem=30000]\" -M 30000000 -a \'docker(scao/dailybox)\' -o $lsf_out -e $lsf_err sh $sh_file\n";
system ( $bsub_com );
}
sub bsub_parsemhc{
my ($step_by_step) = @_;
if ($step_by_step) {
$hold_job_file = "";
}else{
$hold_job_file = $current_job_file;
}
#my $cdhitReport = $sample_full_path."/".$sample_name.".fa.cdhitReport";
$current_job_file = "j6_parsebind_".$sample_name.".sh";
my $lsf_out=$lsf_file_dir."/".$current_job_file.".out";
my $lsf_err=$lsf_file_dir."/".$current_job_file.".err";
`rm $lsf_out`;
`rm $lsf_err`;
#my $IN_sam = $sample_full_path."/".$sample_name.".exome.sam";
open(PMHC, ">$job_files_dir/$current_job_file") or die $!;
print PMHC "#!/bin/bash\n";
# print PMHC "#BSUB -n 1\n";
# print PMHC "#BSUB -R \"rusage[mem=30000]\"","\n";
# print PMHC "#BSUB -M 30000000\n";
# print PMHC "#BSUB -q ding-lab\n";
# print PMHC "#BSUB -o $lsf_file_dir","/","$current_job_file.out\n";
# print PMHC "#BSUB -e $lsf_file_dir","/","$current_job_file.err\n";
# print PMHC "#BSUB -J $current_job_file\n";
# print PMHC "#BSUB -w \"$hold_job_file\"","\n";
print PMHC "f_indel=".$sample_full_path."/$sample_name.indel.vcf\n";
print PMHC "f_snv=".$sample_full_path."/$sample_name.snp.vcf\n";
print PMHC "f_indel_wt_fa=".$sample_full_path."/$sample_name.indel.vcf.proteome-indel-wt.fasta\n";
print PMHC "f_snv_wt_fa=".$sample_full_path."/$sample_name.snp.vcf.proteome-snv-wt.fasta\n";
print PMHC "f_indel_mut_fa=".$sample_full_path."/$sample_name.indel.vcf.proteome-indel-mut.fasta\n";
print PMHC "f_snv_mut_fa=".$sample_full_path."/$sample_name.snp.vcf.proteome-snv-mut.fasta\n";
print PMHC "f_netMHC_result=".$sample_full_path."/netMHC4.0.out.append.txt\n";
print PMHC "f_out=".$sample_full_path."/$sample_name.neoantigen.tsv\n";
print PMHC "f_sum=".$sample_full_path."/$sample_name.neo.summary\n";
print PMHC "f_min=".$sample_full_path."/$sample_name.neo.summary.min\n";
print PMHC " ".$run_script_path_perl."parseNetMHC4result.pl \${f_netMHC_result} \${f_indel_wt_fa} \${f_snv_wt_fa} \${f_out}"."\n";
print PMHC " ".$run_script_path_perl."reportSummary.pl \${f_out} \${f_indel} \${f_snv} \${f_indel_mut_fa} \${f_snv_mut_fa} \${f_sum}"."\n";
print PMHC " ".$run_script_path_perl."get_min_result.pl \${f_sum} \${f_min}"."\n";
print PMHC "rm \${f_netMHC_result}"."\n";
close PMHC;
#$bsub_com = "bsub < $job_files_dir/$current_job_file\n";
#system ( $bsub_com );
my $sh_file=$job_files_dir."/".$current_job_file;
# $bsub_com = "bsub -q research-hpc -n 1 -R \"select[mem>30000] rusage[mem=30000]\" -M 30000000 -a \'docker(registry.gsc.wustl.edu/genome/genome_perl_environment)\' -w $hold_job_file -o $lsf_out -e $lsf_err sh $sh_file\n";
$bsub_com = "bsub -q research-hpc -n 1 -R \"select[mem>30000] rusage[mem=30000]\" -M 30000000 -a \'docker(registry.gsc.wustl.edu/genome/genome_perl_environment)\' -w \"$hold_job_file\" -o $lsf_out -e $lsf_err sh $sh_file\n";
#$bsub_com = "bsub < $job_files_dir/$current_job_file\n";
system ( $bsub_com );
}
sub bsub_get_ref_count()
{
my ($step_by_step) = @_;
if ($step_by_step) {
$hold_job_file = "";
}else{
$hold_job_file = $current_job_file;
}
#my $cdhitReport = $sample_full_path."/".$sample_name.".fa.cdhitReport";
$current_job_file = "j7_getrefcount_".$sample_name.".sh";
my $lsf_out=$lsf_file_dir."/".$current_job_file.".out";
my $lsf_err=$lsf_file_dir."/".$current_job_file.".err";
`rm $lsf_out`;
`rm $lsf_err`;
#my $IN_sam = $sample_full_path."/".$sample_name.".exome.sam";
my $IN_bam = $sample_full_path."/".$sample_name.".rnaseq.bam";
open(REFC, ">$job_files_dir/$current_job_file") or die $!;
print REFC "#!/bin/bash\n";
# print REFC "#BSUB -n 1\n";
# print REFC "#BSUB -R \"rusage[mem=30000]\"","\n";
# print REFC "#BSUB -M 30000000\n";
# print REFC "#BSUB -q ding-lab\n";
# print REFC "#BSUB -o $lsf_file_dir","/","$current_job_file.out\n";
# print REFC "#BSUB -e $lsf_file_dir","/","$current_job_file.err\n";
# print REFC "#BSUB -J $current_job_file\n";
# print REFC "#BSUB -w \"$hold_job_file\"","\n";
print REFC "f_fa_all=".$sample_full_path."/$sample_name.transcript.fa\n";
print REFC " ".$run_script_path_perl."extract_reads_supporting_ref.pl $IN_bam $db_ref_bed \${f_fa_all}"."\n";
close REFC;
# $bsub_com = "bsub < $job_files_dir/$current_job_file\n";
# system ( $bsub_com );
my $sh_file=$job_files_dir."/".$current_job_file;
# $bsub_com = "bsub -q research-hpc -n 1 -R \"select[mem>30000] rusage[mem=30000]\" -M 30000000 -a \'docker(registry.gsc.wustl.edu/genome/genome_perl_environment)\' -w $hold_job_file -o $lsf_out -e $lsf_err sh $sh_file\n";
$bsub_com = "bsub -q research-hpc -n 1 -R \"select[mem>30000] rusage[mem=30000]\" -M 30000000 -a \'docker(registry.gsc.wustl.edu/genome/genome_perl_environment)\' -w \"$hold_job_file\" -o $lsf_out -e $lsf_err sh $sh_file\n";
#$bsub_com = "bsub < $job_files_dir/$current_job_file\n";
system ( $bsub_com );
}
sub bsub_final_report()
{
my ($step_by_step) = @_;
if ($step_by_step) {
$hold_job_file = "";
}else{
$hold_job_file = $current_job_file;
}
#my $cdhitReport = $sample_full_path."/".$sample_name.".fa.cdhitReport";
$current_job_file = "j8_final_report_".$working_name.".sh";
#my $IN_sam = $sample_full_path."/".$sample_name.".exome.sam";
my $lsf_out=$lsf_file_dir."/".$current_job_file.".out";
my $lsf_err=$lsf_file_dir."/".$current_job_file.".err";
`rm $lsf_out`;
`rm $lsf_err`;
open(REP, ">$job_files_dir/$current_job_file") or die $!;
print REP "#!/bin/bash\n";
#print REP "#BSUB -n 1\n";
#print REP "#BSUB -R \"rusage[mem=30000]\"","\n";
#print REP "#BSUB -M 30000000\n";
#print REP "#BSUB -o $lsf_file_dir","/","$current_job_file.out\n";
#print REP "#BSUB -e $lsf_file_dir","/","$current_job_file.err\n";
#print REP "#BSUB -q ding-lab\n";
#print REP "#BSUB -J $current_job_file\n";
#print REP "#BSUB -w \"$hold_job_file\"","\n";
print REP " ".$run_script_path_perl."generate_report_summary.pl $run_dir $version"."\n";
close REP;
#$bsub_com = "bsub < $job_files_dir/$current_job_file\n";
#system ( $bsub_com );
my $sh_file=$job_files_dir."/".$current_job_file;
# $bsub_com = "bsub -q research-hpc -n 1 -R \"select[mem>30000] rusage[mem=30000]\" -M 30000000 -a \'docker(registry.gsc.wustl.edu/genome/genome_perl_environment)\' -w $hold_job_file -o $lsf_out -e $lsf_err sh $sh_file\n";
$bsub_com = "bsub -q research-hpc -n 1 -R \"select[mem>30000] rusage[mem=30000]\" -M 30000000 -a \'docker(registry.gsc.wustl.edu/genome/genome_perl_environment)\' -w \"$hold_job_file\" -o $lsf_out -e $lsf_err sh $sh_file\n";
#$bsub_com = "bsub < $job_files_dir/$current_job_file\n";
system ( $bsub_com );
}