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runPipeline
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#!/usr/bin/env perl
use strict;
use warnings;
use File::Basename;
use Getopt::Long;
use LWP::UserAgent;
use Term::ANSIColor;
use POSIX qw(strftime);
use Cwd;
use threads;
use FindBin qw($RealBin);
use File::Path qw(make_path remove_tree);
use File::Copy;
use lib "$RealBin/lib";
#use File::Tee qw(tee);
require "$RealBin/edge_ui/metadata_scripts/metadata_api.pl";
my $workingDir = Cwd::getcwd();
$ENV{PATH} = "$RealBin:$RealBin/bin/:$RealBin/scripts/:$RealBin/thirdParty/Anaconda2/bin/:$ENV{PATH}:/usr/sbin:/opt/apps/bin";
$ENV{PERL5LIB} = ($ENV{PERL5LIB})? "$RealBin/lib:$RealBin/lib/perl5:$RealBin/lib/perl5/x86_64-linux-thread-multi:$ENV{PERL5LIB}":"$RealBin/lib:$RealBin/lib/perl5:$RealBin/lib/perl5/x86_64-linux-thread-multi";
$ENV{PERL5SHELL} = "bash";
$ENV{EDGE_HOME} = $RealBin;
$ENV{HOME} ||= $RealBin;
$ENV{LANG} ||= "C.UTF-8";
#$ENV{LC_ALL} ||= "C.UTF-8";
# unbuffer
$|=1;
my $version="2.4.0";
my $debug=0;
my @unpairedReadsFile;
my @pairedReadsFile;
my $outDir;
my $numCPU=8;
my $configFile;
my @referenceGenome;
my $inputContigs="";
my $noColorLog=0;
my $primerFile;
my $proxy=$ENV{HTTP_PROXY}||$ENV{http_proxy};
my ($annotationGFF,$annotationFAA,$annotationFNA); #assembly annotation files
my $data_cleanup;
GetOptions("u=s{,}" => \@unpairedReadsFile,
"p=s{,}" => \@pairedReadsFile,
"o=s" => \$outDir,
"ref=s{,}" => \@referenceGenome,
"contigs=s" => \$inputContigs,
"primer=s" => \$primerFile,
"cpu=i" => \$numCPU,
"config|c=s" => \$configFile,
"noColorLog" => \$noColorLog,
"debug" => \$debug,
"data_cleanup" => \$data_cleanup,
"version" => sub{printVersion()},
"tools" => sub{printEDGETools()},
"help|?" => sub{Usage()} );
sub Usage
{
my $msg=shift;
print ("\nfailed ... ".$msg."\n\n") if $msg;
print <<"END";
Usage: perl $0 [options] -c config.txt -p reads1.fastq reads2.fastq -o out_directory
Version $version
Input File:
-u Unpaired reads, Single end reads in fastq
-p Paired reads in two fastq files and separate by space
-contigs Contig Fasta File.
-c Config File
Output:
-o Output directory.
Options:
-ref Reference genome file in fasta
It will find the genbank file (same prefix) in the same location if any.
-primer A pair of Primers sequences in strict fasta format
-cpu number of CPUs (default: 8)
-data_cleanup remove .sam .bam .gz .fastq .fq. tgz files after run finished.
-version print verison
END
exit;
}
&Usage("Missing Configuration File.") unless ($configFile);
## Global hash reference for configurations.
my $configuration=&readEDGEConfig($configFile);
my $sys = &getSysParams("$RealBin/edge_ui/sys.properties");
&Usage("Missing input files.") unless (@unpairedReadsFile or @pairedReadsFile or $configuration->{'DoSRADownload'} or $configuration->{qiime_input_dir} or $configuration->{targetedNGS_input_dir} or $configuration->{inputContigs} or $inputContigs or $configuration->{piret_input_dir});
&Usage("Missing output directory path.") unless ($outDir);
&make_dir($outDir);
my $abs_outDir=Cwd::abs_path("$outDir");
unlink "$abs_outDir/HTML_Report/writeHTMLReport.finished";
&setProxy if $sys->{proxy};
# Capture error log
my $start_time_string=&getTmpNameByTime;
my $process_log_file = "$outDir/process.log";
my $error_log_file="$outDir/error.log";
open ( my $log_fh, ">>", $process_log_file) or die "Failed to write $process_log_file\n$!";
open(STDERR, '>&', STDOUT) or die "Can't redirect stderr: $!";
open(STDERR, '>', $error_log_file) or die "Can't redirect stderr: $!";
$SIG{__WARN__} = sub {print STDERR @_; &lprint (@_)};
$SIG{__DIE__} = sub {print STDERR @_; &lprint (@_);&send_error_notification();exit 1};
# print whole running command and config file to process log
&lprint("\nProject Start: $start_time_string\n");
print $log_fh qx/ps -ww -o args $$/;
&lprint("Version: $version\n");
open (my $config_fh, "$configFile") or die "Cannot open $configFile\n$!";
while(<$config_fh>){ if (! /user|edgeui\_/) {print $log_fh $_;}}
close $config_fh;
if ( ! -e "$outDir/config.txt"){ copy("$configFile","$outDir/config.txt");}
$numCPU = $configuration->{'cpu'} if ($configuration->{'cpu'});
$inputContigs = $configuration->{inputContigs} || "" unless $inputContigs;
if (!@referenceGenome and $configuration->{reference})
{
if (scalar @{$configuration->{reference}}){
@referenceGenome= @{$configuration->{reference}};
}
else{
@referenceGenome= split /,/, $configuration->{reference};
}
}
$primerFile ||= $configuration->{primer};
my $project_name = $configuration->{projname}||basename($outDir);
if (length ($project_name) > 30 && $configuration->{DoAnnotation} && $configuration->{annotateProgram} =~ /prokka/i ){
&lprint("Project Name is too long (> 30 characters) will casue errors for Prokka annotation pipeline");
}
#&checkDependedPrograms;
my $download_sra_retry_limit=3;
my $download_sra_retry=0;
if ($configuration->{'DoSRADownload'})
{
&Download_SRA();
}
if ($configuration->{qiime_input_dir} || $configuration->{targetedNGS_input_dir} || $configuration->{piret_input_dir}){
my $dir_input=$configuration->{qiime_input_dir} || $configuration->{targetedNGS_input_dir} || $configuration->{piret_input_dir};
my $mapping_files=$configuration->{qiime_mapping_file} || $configuration->{targetedNGS_sample_file} || $configuration->{piret_exp_design_file};
my ($pe_files_r,$se_files_r)=&parse_qiime_mapping_file($dir_input,$mapping_files);
@pairedReadsFile=@{$pe_files_r};
@unpairedReadsFile=@{$se_files_r};
}
@pairedReadsFile = &checkFileAndDir;
my $count_file_list= "$outDir/QcReads/fastqCount.txt";
my ($QCpairFile,$QCunpairFile);
if(@unpairedReadsFile or @pairedReadsFile)
{
($QCpairFile,$QCunpairFile)=&countFastq(\@unpairedReadsFile, \@pairedReadsFile);
}
if ($configuration->{DoQC})
{
($QCpairFile,$QCunpairFile)=&runQC($QCpairFile,$QCunpairFile);
}
if ($configuration->{DoJoinPE}){
($QCpairFile,$QCunpairFile)=&runJoinPE($QCpairFile,$QCunpairFile);
}
if ($configuration->{'DoQiimeAnalysis'}){
&runQiimeAnalysis(\@unpairedReadsFile, \@pairedReadsFile);
}
if ($configuration->{DoDETEQT}){
&runDETEQT();
}
if ($configuration->{DoPiReTAnalysis}){
&runPiReT();
}
if ($configuration->{DoHostRemoval})
{
my $time=time();
my @host_files = @{$configuration->{Host}};
foreach my $host_file (@{$configuration->{Host}})
{
($QCpairFile,$QCunpairFile)=&runHostRemoval($QCpairFile,$QCunpairFile,$host_file);
}
&runHostRemovalStat();
$QCpairFile = "$outDir/HostRemoval/hostclean.1.fastq $outDir/HostRemoval/hostclean.2.fastq" if ($QCpairFile);
$QCunpairFile = "$outDir/HostRemoval/hostclean.unpaired.fastq" if ($QCunpairFile);
&printRunTime($time) if ( ! -e "$outDir/HostRemoval/HostRemovalStats.pdf");
}
my $contigFile="";
my $contig_for_annotation_file;
my $contig_size_for_annotation=$configuration->{contig_size_cut_for_annotation}||700;
if ($configuration->{assembledContigs} || $inputContigs){
($contigFile,$contig_for_annotation_file)=&processProvideContigs();
}
if ($configuration->{DoAssembly} && ($QCpairFile or $QCunpairFile))
{
my $assembler = $configuration->{assembler};
($contigFile,$contig_for_annotation_file)=&runAssembly($QCpairFile,$QCunpairFile,$assembler) if (!$configuration->{assembledContigs});
&runReadsToContig($QCpairFile,$QCunpairFile,$contigFile) if ($contigFile and $configuration->{DoReadsMappingContigs});
}
my $tax_kingdom=$configuration->{kingdom};
my $referenceGenome;
my $genbankFile;
my $gff3File;
my $ref_headers;#array ref
my $annotateProgram=$configuration->{annotateProgram}||"prokka";
($referenceGenome,$genbankFile,$gff3File, $ref_headers)=&check_reference_genome(\@referenceGenome) if (@referenceGenome);
if ($referenceGenome)
{
my ($r2gVCF,$r2gGap);
my ($r2gUnmappedPaired,$r2gUnmappedSingle,$numOfunmappedReads);
if ($QCpairFile or $QCunpairFile){
if ($configuration->{DoReadsMappingReference}){
($r2gVCF,$r2gGap)=&runReadsToGenome($QCpairFile,$QCunpairFile,\@referenceGenome, $ref_headers);
if ($configuration->{MapUnmappedReads} || $configuration->{r2g_extract_unmapped}){
($r2gUnmappedPaired, $r2gUnmappedSingle,$numOfunmappedReads) = &retrieveUnmappedReads(\@referenceGenome);
}
if( $configuration->{MapUnmappedReads} ){
&runUnmappedReadsToRefSeq($r2gUnmappedPaired, $r2gUnmappedSingle) if ($numOfunmappedReads > 0);
}
}
# taxonomy analysis
if ($configuration->{DoReadsTaxonomy})
{
if ($configuration->{AllReads})
{
&Reads_taxonomyAssignment($QCpairFile,$QCunpairFile,"$outDir/ReadsBasedAnalysis/Taxonomy");
}
else
{
# retrieve unmapped reads
($r2gUnmappedPaired,$r2gUnmappedSingle,$numOfunmappedReads) = &retrieveUnmappedReads(\@referenceGenome);
&Reads_taxonomyAssignment($r2gUnmappedPaired,$r2gUnmappedSingle,"$outDir/ReadsBasedAnalysis/UnmappedReads/Taxonomy") if ($numOfunmappedReads > 0);
symlink("$outDir/ReferenceBasedAnalysis/UnmappedReads", "$outDir/ReadsBasedAnalysis/UnmappedReads") if ( ! -e "$outDir/ReadsBasedAnalysis/UnmappedReads");
}
}
}
my ($unusedContigFasta,$c2g_SNP_INDEL_file,$c2g_Gap);
if ( $contigFile && -s $contigFile)
{
($unusedContigFasta,$c2g_SNP_INDEL_file,$c2g_Gap)=&runContigToGenome($contigFile,$referenceGenome);
#blast nr on novel contigs/regions
#&ContigsBlast($unusedContigFasta,'nt') if ($configuration->{DoBlast});
#`ln -sf $outDir/AssemblyBasedAnalysis/Blast $outDir/ReferenceBasedAnalysis/UnmapppedContigs`;
&runUnmappedContigToRefSeq($unusedContigFasta) if $configuration->{MapUnmappedContigs} && -s $unusedContigFasta;
}
#SNP/INDEL analysis
&variantAnalysis($c2g_SNP_INDEL_file,$c2g_Gap,$r2gVCF,$r2gGap,$gff3File, $referenceGenome) if ($configuration->{DoVariantAnalysis});
}
else # reference unknown
{
if ($QCpairFile or $QCunpairFile){
# Reads taxonomy
&Reads_taxonomyAssignment($QCpairFile,$QCunpairFile,"$outDir/ReadsBasedAnalysis/Taxonomy") if ($configuration->{DoReadsTaxonomy});
}
#blast nt for contigs taxonomy
&ContigsBlast($contigFile,'nt') if ($configuration->{DoBlast});
}
if ($configuration->{DoContigsTaxonomy} and $contigFile)
{
&Contigs_taxonomyAssignment($contigFile);
}
if ( $contigFile && -s $contigFile )
{
#not sure if it is Virus, use size of references to determine the kingdoma (580K is smallest Prok,Mycoplasma)
my $contigTotalSize=0;
$contigTotalSize = `grep ${project_name}_contigs.fa $outDir/AssemblyBasedAnalysis/contigs_stats.txt | awk \'{print \$9}\'` if (-e "$outDir/AssemblyBasedAnalysis/contigs_stats.txt");
if (!$tax_kingdom)
{
$tax_kingdom = ($contigTotalSize >= 580000) ? "Bacteria":"Viruses";
}
if ($configuration->{DoAnnotation})
{
($annotationGFF,$annotationFAA,$annotationFNA)=&runAnnotation($contig_for_annotation_file,$tax_kingdom,$annotateProgram,$project_name);
#phagefinder if Bacteria
&phageFinder($annotationGFF,$annotationFAA,$annotationFNA) if ($tax_kingdom ne "Viruses" and $configuration->{DoProPhageDetection});
}
if ($configuration->{DoSMA}){ #Secondary Metabolite Analysis
my $input_for_antismash = ( -e "$outDir/AssemblyBasedAnalysis/Annotation/$project_name.gbk")? "$outDir/AssemblyBasedAnalysis/Annotation/$project_name.gbk" : $contigFile;
&runAntiSmash($input_for_antismash);
}
if ($configuration->{DoBinning}){
&runBinning($contigFile);
if ($configuration->{DoCheckM}){
&runCheckM("$outDir/AssemblyBasedAnalysis/Binning");
}
}
}
if ($configuration->{DoSNPtree})
{
&SNPtree($QCpairFile,$QCunpairFile,$contigFile,$tax_kingdom);
}
if ($configuration->{DoReadsSpecialtyGenes} || $configuration->{DoORFsSpecialtyGenes})
{
&runSpecialtyGenesProfiling($QCpairFile,$QCunpairFile,$annotationFAA,$annotationGFF);
}
my $pcrCheckFailure=0;
if ($primerFile and $configuration->{DoPrimerValidation})
{
$pcrCheckFailure=&pcrValidation($primerFile,$contigFile, $referenceGenome);
}
if ( $contigFile && -s $contigFile)
{
#primer Design for Assay Failure and Novel pathgen
if ($pcrCheckFailure == 1 || $configuration->{DoPrimerDesign})
{
&pcrDesign($contigFile);
}
}
# generate JBrowse Tracks
&writeJBrowseInfo($outDir) if ($configuration->{DoJBrowse});
my $taxonomy_report_dir;
my $conversion_cmd_array_ref = &MergeReport();
#&lprint ("All Done. \n See $final_report for the Final pdf Report\n");
&generateHTMLreport($outDir,$conversion_cmd_array_ref) if ($configuration->{DoHTMLReport});
#&updateProjectHTML($outDir,$start_time_string) if ($configuration->{DoHTMLReport});
if ($configuration->{DoReadsTaxonomy} && $configuration->{enabledTools} =~ /gottcha/){
##pathogen detection
my $blist = "$taxonomy_report_dir/1_allReads/gottcha2-speDB-b/allReads-gottcha2-speDB-b.list.txt";
my $vlist = "$taxonomy_report_dir/1_allReads/gottcha2-speDB-v/allReads-gottcha2-speDB-v.list.txt";
if(!-e $blist) {
$blist = "$taxonomy_report_dir/1_allReads/gottcha-speDB-b/allReads-gottcha-speDB-b.list.txt";
}
if(!-e $vlist) {
$vlist = "$taxonomy_report_dir/1_allReads/gottcha-speDB-v/allReads-gottcha-speDB-v.list.txt";
}
if($sys->{edge_pathogen_detection} && -e $blist && -e $vlist) {
&detectPathogen($blist, $vlist);
}elsif($sys->{edge_pathogen_detection}){
&lprint ("File $blist not found\n");
&lprint ("File $vlist not found\n");
}
}
##END pathogen detection
##sample metadata submission
if($sys->{edge_sample_metadata_autosubmit2bsve}) {
&lprint ("\nSubmit metadata to bsve ...");
&pushSampleMetadata("add", $outDir, $sys);
&lprint ("Metadata finished.\n\n");
}
##END sample metadata
&lprint ("Total");
my $total_run_time=&printRunTime($^T);
&lprint ("All Done. \n\n");
close $log_fh;
`echo "$start_time_string\t$total_run_time" >> $abs_outDir/.AllDone`;
my $mail_subject= "EDGE project $project_name complete";
my $recipient = $configuration->{projowner};
$recipient = "$recipient,$sys->{email_recipient}" if ($sys->{email_recipient});
my $projID= $configuration->{projcode} || $configuration->{projid};
my $mail_msg = "EDGE project $project_name (Description: $configuration->{projdesc}) is complete. Please login to ".
"$configuration->{projrunhost}/ to see the result.".
"\nOr click link below\n\n $configuration->{projrunhost}/?proj=$projID\n";
&sendMail($sys->{cluster_job_notify},$recipient,$mail_subject,$mail_msg) if ($sys->{email_notification} && $recipient);
## DATA Clean up ##
if ($configuration->{projrunhost} =~ /edge-bsve/ || defined $data_cleanup ){
my $cmd = "$RealBin/scripts/data_cleanup.pl $abs_outDir";
system($cmd);
}
unlink glob("$ENV{HOME}/core.*");
###
#my $url = "http://localhost";
#my $pid=fork();
#if ($pid == 0)
#{
# unless ( &check_server_up($url) || &check_server_up("$url:8080") )
# {
#system ("$RealBin/scripts/httpserver.py --webdir $RealBin/edge_ui & ");
# &open_default_browser("file:///$abs_outDir/HTML_Report/report.html");
# }
#exit 0;
#}
### END MAIN ###
### Below are subroutins ###
sub runPiReT {
my $exp_file = $configuration->{piret_exp_design_file};
my $piret_indir = $configuration->{piret_input_dir};
my $outputDir = "$outDir/ReferenceBasedAnalysis/Piret";
my $exp_design = "$outputDir/exp_design.txt";
my $log = "$outputDir/piret_log.txt";
my $luigi_config = "$outputDir/luigi.cfg";
my $run_script = "$outputDir/runPiret.sh";
my $time=time();
if ($noColorLog)
{
&lprint ("[PiReT transcriptomics analysis]\n");
}
else
{
&lprint (colored ("[PiReT transcriptomics analysis]\n",'yellow'));
}
if ( -e "$outputDir/runPiret.finished" )
{
&lprint ("PiReT transcriptomics analysis Finished\n");
return 0;
}
if (! -e $exp_file) {
&lprint("ERROR: No experimental design file");
exit;
}
make_path($outputDir,{chmod => 0755,});
my $piret_indir_basename=basename($piret_indir);
my $piret_abs_indir=Cwd::abs_path($piret_indir);
symlink($piret_abs_indir,"$outputDir/$piret_indir_basename");
&prepareExpDesignAndConfig($exp_file,$piret_indir_basename,$outputDir);
chdir "$outputDir";
my $cmd="$RealBin/thirdParty/piret/bin/piret -e $exp_design -d $outputDir --config $luigi_config 2>$log";
&lprint (" Running \n $cmd \n");
open (my $fh, ">", $run_script);
my $piret_env_path1 = "$RealBin/thirdParty/piret/thirdParty/miniconda/envs/piret";
my $piret_env_path2 = "$RealBin/thirdParty/Anaconda3/envs/piret";
my $piret_env = (-e $piret_env_path1)? $piret_env_path1 : ( -e $piret_env_path2)? $piret_env_path2 : "piret";
print $fh "source $RealBin/thirdParty/Anaconda3/bin/activate $piret_env 1>/dev/null\n";
print $fh "LUIGI_CONFIG_PATH=$luigi_config ";
print $fh $cmd,"\n";
#print $fh "source $RealBin/thirdParty/Anaconda3/bin/deactivate piret 1>/dev/null\n";
close $fh;
&executeCommand2("/bin/bash $run_script");
#unlink "$outputDir/$piret_indir_basename";
&touchFile("$outputDir/runPiret.finished");
&printRunTime($time);
return 0;
}
sub prepareExpDesignAndConfig{
my $exp_file = shift;
my $dir= shift;
my $outdir=shift;
my $exp_design = "$outdir/exp_design.txt";
my $luigi_config = "$outdir/luigi.cfg";
my $stranded = ($configuration->{piret_stranded})?$configuration->{piret_stranded}:0;
my $kingdom = $configuration->{piret_kingdom};
my $test_method = $configuration->{piret_method}||'DEedge';
my $significant_pvalue = $configuration->{piret_p_value} || 0.001;
my $prokaryote_fasta = ($configuration->{piret_prokaryote_fasta})?$configuration->{piret_prokaryote_fasta}:"NA";
my $prokaryote_gff = ($configuration->{piret_prokaryote_gff})?$configuration->{piret_prokaryote_gff}:"NA";
my $eukarya_fasta = ($configuration->{piret_eukarya_fasta})?$configuration->{piret_eukarya_fasta}:"NA";
my $eukarya_gff = ($configuration->{piret_eukarya_gff})?$configuration->{piret_eukarya_gff}:"NA";
my $index_ref = $configuration->{piret_index} || "hisat_index";
my $fh;
my $method_config="edgeR, DESeq2";
if ($test_method eq "edgeR"){
$method_config = "edgeR";
}elsif($test_method eq "DESeq2"){
$method_config = "DESeq2";
}elsif($test_method eq "DEedge"){
$method_config = "edgeR, DESeq2";
}elsif($test_method eq "ballgown"){
$method_config = "ballgown";
}elsif($test_method eq "balledgeR"){
$method_config = "ballgown, edgeR";
}elsif($test_method eq "DEgown"){
$method_config = "DESeq2, ballgown";
}
if ($kingdom eq 'prokarya')
{
if ( !-e $prokaryote_fasta || ! -e $prokaryote_gff){
&lprint("ERROR: Please provide prokaryote fasta and gff files");
exit;
}
if (! is_gff($prokaryote_gff)){
&lprint("ERROR:prokaryote_gff is not in GFF3 format");
exit;
}
}
elsif ($kingdom eq 'eukarya')
{
if ( ! -e $eukarya_fasta || ! -e $eukarya_gff){
&lprint("ERROR: Please provide eukaryote fasta and gff files");
exit;
}
if (! is_gff($eukarya_gff)){
&lprint("ERROR:eukarya_gff is not in GFF3 format");
exit;
}
}
elsif ($kingdom eq 'both')
{
if ( !-e $prokaryote_fasta || ! -e $prokaryote_gff){
&lprint("ERROR: Please provide prokaryote fasta and gff files");
exit;
}
if ( ! -e $eukarya_fasta || ! -e $eukarya_gff){
&lprint("ERROR: Please provide eukaryote fasta and gff files");
exit;
}
if (! is_gff($prokaryote_gff) || ! is_gff($eukarya_gff)){
&lprint("ERROR:prokaryote_gff or eukarya_gff is not in GFF3 format");
exit;
}
}
if ($exp_file =~ /xlsx$/){
open ($fh, "-|")
or exec("xlsx2csv", "-d", "tab", "$exp_file");
}else{
open $fh,"<", $exp_file;
}
open my $ofh, ">", $exp_design;
my $file_column_index;
while(<$fh>){
chomp;
next if (/^\n/);
next unless (/\S/);
if (/ID/ && /Files/i){
my @header = split /\t/,$_;
( $file_column_index )= grep { $header[$_] =~ /files/i } 0..$#header;
print $ofh $_,"\n";
}elsif(! /^#/){
my @array = split /\t/,$_;
$array[$file_column_index] =~ tr/"//d;
$array[$file_column_index] =~ s/^\s+|\s+$//g;
$array[$file_column_index] =~ s/:/:$dir\//;
$array[$file_column_index] = "$dir/$array[$file_column_index]";
$array[0] =~ s/[^\w-]/_/g;
print $ofh join("\t",@array),"\n";
}
}
close $fh;
close $ofh;
my $log_level = ($debug)? "DEBUG":"INFO";
open (my $ofh2, ">",$luigi_config);
print $ofh2 <<"END";
[DEFAULT]
QC=True
#are the libraries stranded
stranded=$stranded
#Which alignment to use, star or hisat2
aligner=hisat2
#is it prokaryotic or eukaryotic organism, or has both, affects isoforms
kingdom=$kingdom
threads=$numCPU
#use one or all the method to find DEG, edgeR, DESeq2, ballgown
method=[$method_config]
#select a qvalue to declare a gene DEG, corresponds to p-value after multiple test corrections
q_value=$significant_pvalue
#Do you want to find novel non-coding regions?
novel=True
fasta_prok=$prokaryote_fasta
fasta_euk=$eukarya_fasta
gff_prok=$prokaryote_gff
gff_euk=$eukarya_gff
#do you want it to find pathways assocaited with your DEGs
pathway=True
#gene enrichment analysis using GO#s
gage=True
hisat_index=$index_ref
star_index=star_index
# full path to emap database
emap_db=$RealBin/database/EGGNOG
[core]
log_level:$log_level
[RunAllQC]
[SummarizeQC]
faqc_min_L=50
n_cutoff=4
avg_q=5
#-----------------------------------Mapping step -----------------------------#
## HISAT options
## hisat2 index generation options
[HisatIndex]
[Hisat]
# minimum intron length (20)
min_introlen=20
# # maximum intro length
max_introlen=500000
# # strandness information, if its F, R, FR, or FR
rna_strandness=FR
[HisatMapW]
[STARindex]
# STAR options
[map_star]
align_intron_min=21
align_intron_max=0
[map_starW]
[ExtractPP]
[ExtractPPW]
[FindNovelRegions]
[FindNovelRegionsW]
[CompileGFF]
[RefNames]
[GetRefNames]
[SplitBAMfile]
[SplitBAMBoth]
[Split2ProkEuk]
[Split2ProkEukW]
[StringTieScores]
[StringTieScoresW]
[FeatureCounts]
fid=ID
stranded=$stranded
[FeatureCountsBoth]
fid=ID
stranded=$stranded
[MergeStringTies]
[ReStringTieScoresW]
[ReStringTieScores]
[RunEmapper]
query_coverage=0.0
subject_coverage=0.0
ave_map=100
[edgeR]
fid=ID
[DESeq2]
fid=ID
END
close $ofh2;
return 0;
}
sub runSpecialtyGenesProfiling {
my $pair_read = shift;
my $single_end_read= shift;
my $annotationFAA = shift;
my $annotationGFF = shift;
my $search_tool = $configuration->{SpecialtyGenesSearchTool} || "rapsearch2";
my $ShortBRED_PATH = "$RealBin/bin/ShortBRED";
#my $rgi_PATH = "$RealBin/thirdParty/Anaconda3/bin";
my $cov_table="$abs_outDir/AssemblyBasedAnalysis/readsMappingToContig/readsToContigs_coverage.table";
my $identity = $configuration->{ShortBREDMinPercIdentity} || "0.95";
my $pctlength = $configuration->{ShortBREDMinPercLength} || "0.95";
my $time=time();
my $CARD_json = "$RealBin/database/ShortBRED/AR/card.json";
my $VF_markers = "$RealBin/database/ShortBRED/VF/VFDB_9aa_markers.faa";
my $AR_markers = "$RealBin/database/ShortBRED/AR/ShortBRED_ABR_101bp_markers.faa";
my $AR_metadata = "$RealBin/database/ShortBRED/AR/ShortBRED_ABR_Metadata.tab";
my $specialtyGenesDone = 0;
my $VFDB_ini="$RealBin/scripts/specialty_genes/mysql.ini";
if ($noColorLog)
{
&lprint ("[Gene Family Analysis]\n");
}
else
{
&lprint (colored ("[Gene Family Analysis]\n",'yellow'));
}
if ($configuration->{DoReadsSpecialtyGenes} and $configuration->{DoORFsSpecialtyGenes})
{
if (-e "$outDir/ReadsBasedAnalysis/SpecialtyGenes/runSpecialtyGenesProfiling.finished" and -e "$outDir/AssemblyBasedAnalysis/SpecialtyGenes/runSpecialtyGenesProfiling.finished")
{
$specialtyGenesDone = 1;
}
}
elsif ($configuration->{DoReadsSpecialtyGenes} and -e "$outDir/ReadsBasedAnalysis/SpecialtyGenes/runSpecialtyGenesProfiling.finished")
{
$specialtyGenesDone = 1;
}
elsif ($configuration->{DoORFsSpecialtyGenes} and -e "$outDir/AssemblyBasedAnalysis/SpecialtyGenes/runSpecialtyGenesProfiling.finished")
{
$specialtyGenesDone = 1;
}
if ($specialtyGenesDone)
{
&lprint ("Gene Family Analysis Finished\n");
return;
}
unlink "$outDir/JBrowse/writeJBrowseInfo.finished";
my $wgs_reads = "$pair_read " if ($pair_read);
$wgs_reads .= "$single_end_read " if ( $single_end_read && -s $single_end_read);
if ( !-e "$VFDB_ini"){
open (my $fh_ini,">$VFDB_ini") or die "Cannot write to $VFDB_ini\n";
print $fh_ini "[LOGIN]\n";
print $fh_ini "username=".$sys->{VFDB_dbuser}."\n";
print $fh_ini "password=".$sys->{VFDB_dbpasswd}."\n";
print $fh_ini "host=".$sys->{VFDB_dbhost}."\n";
print $fh_ini "port=".$sys->{VFDB_dbport}."\n";
print $fh_ini "database=".$sys->{VFDB_dbname}."\n";
close $fh_ini;
}
# Reads Based Analysis
if ($configuration->{DoReadsSpecialtyGenes} and $wgs_reads and ! -e "$outDir/ReadsBasedAnalysis/SpecialtyGenes/runSpecialtyGenesProfiling.finished")
{
my $outputDir= "$outDir/ReadsBasedAnalysis/SpecialtyGenes";
my $log = "$outputDir/log.txt";
my $cmd;
&make_dir($outputDir);
# AR Reads ShortBRED
$cmd = "$ShortBRED_PATH/shortbred_quantify.py --wgs $wgs_reads --markers $AR_markers --results $outputDir/${project_name}_AR_genes_ShortBRED.txt --tmp $outputDir/${project_name}_AR_genes_tmp --marker_results $outputDir/${project_name}_AR_genes_markers.txt --SBhits $outputDir/${project_name}_AR_genes_SBhits.txt --search_program $search_tool --id $identity --pctlength $pctlength --threads $numCPU 1>$log 2>&1";
&lprint (" Running \n $cmd \n");
&executeCommand($cmd);
$cmd="$RealBin/scripts/specialty_genes/ShortBred_report.pl -SBresult $outputDir/${project_name}_AR_genes_ShortBRED.txt -metadata $AR_metadata -category AR -project $project_name -out $outputDir";
&lprint (" Running \n $cmd \n");
&executeCommand($cmd);
#VF
$cmd = "$ShortBRED_PATH/shortbred_quantify.py --wgs $wgs_reads --markers $VF_markers --results $outputDir/${project_name}_VF_genes_ShortBRED.txt --tmp $outputDir/${project_name}_VF_genes_tmp --marker_results $outputDir/${project_name}_VF_genes_markers.txt --SBhits $outputDir/${project_name}_VF_genes_SBhits.txt --search_program $search_tool --id $identity --pctlength $pctlength --threads $numCPU 1>>$log 2>&1";
&lprint (" Running \n $cmd \n");
&executeCommand($cmd);
$cmd = "python $RealBin/scripts/specialty_genes/ProcessVFSBResults.py -t reads -m $VFDB_ini -i $outputDir/${project_name}_VF_genes_ShortBRED.txt -p $outputDir/${project_name}_VF_genes_ShortBRED 1>>$log 2>&1";
&lprint (" Running \n $cmd \n");
&executeCommand($cmd);
&touchFile("$outputDir/runSpecialtyGenesProfiling.finished");
}
# ORF Based Analysis
if ($configuration->{DoORFsSpecialtyGenes} and -s $annotationFAA and -s $annotationGFF and ! -e "$outDir/AssemblyBasedAnalysis/SpecialtyGenes/runSpecialtyGenesProfiling.finished")
{
my $outputDir= "$outDir/AssemblyBasedAnalysis/SpecialtyGenes";
my $log = "$outputDir/log.txt";
my $cmd;
&make_dir($outputDir);
# AR
my $rgi_env = "$RealBin/thirdParty/Anaconda3/envs/py36";
my $rgi_env_activate_cmd="source $RealBin/thirdParty/Anaconda3/bin/activate $rgi_env 1>/dev/null";
my $deactivate_cmd="source deactivate 2>/dev/null || true";
$cmd = "cd $outputDir; $rgi_env_activate_cmd; rgi load -i $CARD_json --local 1>>$log 2>&1 ; $deactivate_cmd";
&executeCommand($cmd);
$cmd = "cd $outputDir; $rgi_env_activate_cmd; rgi main -i $annotationFAA -t protein -o ${project_name}_AR_genes_rgi -n $numCPU --local 1>>$log 2>&1 ; $deactivate_cmd";
&lprint (" Running \n $cmd \n");
&executeCommand($cmd);
$cmd = "python $RealBin/scripts/specialty_genes/ProcessARRGIJson.py -i $outputDir/${project_name}_AR_genes_rgi.json -g $annotationGFF -p $outputDir/${project_name}_AR_genes_rgi 1>>$log 2>&1";
&lprint (" Running \n $cmd \n");
&executeCommand($cmd);
$cmd = "python $RealBin/scripts/specialty_genes/gff_to_faa.py $annotationFAA $outputDir/${project_name}_AR_genes_rgi.gff > $outputDir/${project_name}_AR_genes_rgi.faa";
&executeCommand($cmd) if ( -s "$outputDir/${project_name}_AR_genes_rgi.gff");
# VF
$cmd = "$ShortBRED_PATH/shortbred_quantify.py --genome $annotationFAA --markers $VF_markers --results $outputDir/${project_name}_VF_genes_ShortBRED.txt --tmp $outputDir/${project_name}_VF_genes_tmp --marker_results $outputDir/${project_name}_VF_genes_markers.txt --SBhits $outputDir/${project_name}_VF_genes_SBhits.txt --search_program $search_tool --id $identity --threads $numCPU 1>>$log 2>&1";
&lprint (" Running \n $cmd \n");
&executeCommand($cmd);
$cmd = "python $RealBin/scripts/specialty_genes/ProcessVFSBResults.py -t orf -m $VFDB_ini -i $outputDir/${project_name}_VF_genes_ShortBRED.txt -s $outputDir/${project_name}_VF_genes_SBhits.txt -g $annotationGFF -p $outputDir/${project_name}_VF_genes_ShortBRED 1>>$log 2>&1";
&lprint (" Running \n $cmd \n");
&executeCommand($cmd);
$cmd = "python $RealBin/scripts/specialty_genes/gff_to_faa.py $annotationFAA $outputDir/${project_name}_VF_genes_ShortBRED.gff > $outputDir/${project_name}_VF_genes_ShortBRED.faa";
&executeCommand($cmd) if (-s "$outputDir/${project_name}_VF_genes_ShortBRED.gff");
&touchFile("$outputDir/runSpecialtyGenesProfiling.finished");
}
&printRunTime($time);
return 0;
}
sub parse_qiime_mapping_file{
my $qiime_dir=shift;
my $mapping_files=shift;
my @unpariedFiles;
my @pairedFiles;
if ( ! -d "$qiime_dir" ){
my $msg = "ERROR: the input $qiime_dir directroy does not exist or isn't a directory\n";
exit(1);
}
my $header_flag=0;
foreach my $f (split /,/,$mapping_files){
my $file_column_index;
my $fh;
if ($f =~ /xlsx$/){
open ($fh, "-|")
or exec("xlsx2csv", "-d", "tab", "$f");
}else{
open ($fh, "<", $f) or die "Cannot read $f\n";
}
while(<$fh>){
chomp;
next if (/^\n/);
next unless (/\S/);
if (/SampleID|ID/){
$header_flag=1;
my @header = split /\t/,$_;
( $file_column_index )= grep { $header[$_] =~ /files/i } 0..$#header;
}elsif(! /^#/){
my @array = split /\t/,$_;
$array[$file_column_index] =~ tr/"//d;
$array[$file_column_index] =~ s/^\s+|\s+$//g;
$array[$file_column_index] =~ s/\s*,\s*/,/g;
my @files = map { "$qiime_dir/$_" } split /,|\s+|:/,$array[$file_column_index];
if (scalar(@files) % 2){
push @unpariedFiles,@files;
}else{
push @pairedFiles,@files;
}
}
}
close $fh;
die "Incorrect metadata mapping file\n" if (!$header_flag);
}
return (\@pairedFiles,\@unpariedFiles);
}
sub runDETEQT{
my $time=time();
my $outputDir="$outDir/DETEQT";
my $log="$outputDir/log.txt";
my $stats_report = "$outputDir/stats/$project_name.report.txt";
my $run_stats = "$outputDir/stats/$project_name.run_stats.txt";
my $mapping_stats = "$outputDir/stats/$project_name.mapping_stats.txt";
if ($noColorLog)
{
&lprint ("[DETEQT analysis]\n");
}
else
{
&lprint (colored ("[DETEQT analysis]\n",'yellow'));
}
if ( -e "$outputDir/runDETEQT.finished" )
{
&lprint ("DETEQT analysis Finished\n");
return 0;
}
&make_dir($outputDir);
my $targetedNGS_ref=$configuration->{targetedNGS_ref};
my $targetedNGS_mode=$configuration->{targetedNGS_mode} || "PE";
my $targetedNGS_platform=$configuration->{targetedNGS_platform} || "illumina";
my $targetedNGS_len_cutoff=$configuration->{targetedNGS_len_cutoff} || 100;
my $targetedNGS_input_dir=$configuration->{targetedNGS_input_dir};
my $targetedNGS_sample_file=$configuration->{targetedNGS_sample_file};
my $targetedNGS_q_cutoff=$configuration->{targetedNGS_q_cutoff} || 0.8145 ;
my $targetedNGS_depth_cutoff=$configuration->{targetedNGS_depth_cutoff} || 1000;
my $targetedNGS_expectedCoverage=$configuration->{targetedNGS_expectedCoverage};
my $targetedNGS_expectedIdentity=$configuration->{targetedNGS_expectedIdentity};
my $targetedNGS_expectedBaseQ=$configuration->{targetedNGS_expectedBaseQ};
my $targetedNGS_expectedMapQ=$configuration->{targetedNGS_expectedMapQ};
my $targetedNGS_coverageWeight=$configuration->{targetedNGS_coverageWeight};
my $targetedNGS_identityWeight=$configuration->{targetedNGS_identityWeight};
my $targetedNGS_baseqWeight=$configuration->{targetedNGS_baseqWeight};
my $targetedNGS_mapqWeight=$configuration->{targetedNGS_mapqWeight};
if (!$targetedNGS_sample_file){
&lprint("ERROR: targetedNGS analysis No sample file");
exit;
}
if (!$targetedNGS_ref || !is_fasta($targetedNGS_ref)){
&lprint("ERROR: targetedNGS analysis No reference file");
exit;
}
if (!$targetedNGS_input_dir){
&lprint("ERROR: targetedNGS analysis No input directory");
exit;
}
my $cmd = "$RealBin/bin/DETEQT/DETEQT --ref $targetedNGS_ref --indir $targetedNGS_input_dir --samples $targetedNGS_sample_file --cpus $numCPU --outdir $outputDir --prefix $project_name --force " ;
$cmd .= "--q_cutoff $targetedNGS_q_cutoff ";
$cmd .= "--depth_cutoff $targetedNGS_depth_cutoff ";
$cmd .= "--len_cutoff $targetedNGS_len_cutoff ";
$cmd .= "--mode $targetedNGS_mode ";
$cmd .= "--aligner minimap2 " if ($targetedNGS_platform =~ /ont2d/);
$cmd .= "--align_options \'-x pacbio\' " if ($targetedNGS_platform =~ /pacbio/);
$cmd .= "--expectedCoverage $targetedNGS_expectedCoverage " if ($targetedNGS_expectedCoverage);
$cmd .= "--expectedIdentity $targetedNGS_expectedIdentity " if ($targetedNGS_expectedIdentity);
$cmd .= "--expectedBaseQ $targetedNGS_expectedBaseQ " if ($targetedNGS_expectedBaseQ);
$cmd .= "--expectedMapQ $targetedNGS_expectedMapQ " if ($targetedNGS_expectedMapQ);
$cmd .= "--coverageWeight $targetedNGS_coverageWeight " if ($targetedNGS_coverageWeight);
$cmd .= "--identityWeight $targetedNGS_identityWeight " if ($targetedNGS_identityWeight);
$cmd .= "--baseqWeight $targetedNGS_baseqWeight " if ($targetedNGS_baseqWeight);
$cmd .= "--mapqWeight $targetedNGS_mapqWeight " if ($targetedNGS_mapqWeight);
$cmd .= "1>$log 2>&1";
&lprint (" Running \n $cmd \n");
&executeCommand($cmd);
# covert tab file to json for GUI display
&executeCommand("perl $RealBin/scripts/tab2Json_for_dataTable.pl -project_dir $abs_outDir $stats_report > $stats_report.json");
&executeCommand("perl $RealBin/scripts/tab2Json_for_dataTable.pl -project_dir $abs_outDir $run_stats > $run_stats.json");
&executeCommand("perl $RealBin/scripts/tab2Json_for_dataTable.pl -project_dir $abs_outDir $mapping_stats > $mapping_stats.json");
my $run_time = &printRunTime($time);
&touchFile("$outputDir/runDETEQT.finished");
return 0;
}
sub runJoinPE{
my $pairedFile=shift;
my $unpairedFile=shift;
my $time=time();
my $outputDir="$outDir/QcReads";
my $log="$outputDir/JoinPE.log";
my $maxDiffPerc = $configuration->{"fastqjoin-maxdiff"} || 8;
my $minOverlap = $configuration->{"fastqjoin-minoverlap"} || 6;
my $useJoinedPEonly = ($configuration->{"fastqjoin-usejoined-only"})? $configuration->{"fastqjoin-usejoined-only"} : "0";
&make_dir($outputDir);
if ($noColorLog)
{
&lprint ("[Join PE Reads]\n");
}
else
{
&lprint (colored ("[Join PE Reads]\n",'yellow'));
}
if (! $pairedFile){
&lprint(" No Paired End Reads. Skip\n");
return($pairedFile,$unpairedFile);
}
if ( -e "$outputDir/reads.join.fastq" && -e "$outputDir/runJoinPE.finished"){
&lprint("Join PE Reads Finished\n");
if ($useJoinedPEonly){
return ("","$unpairedFile");
}else{
return ("$outputDir/reads.un1.fastq $outputDir/reads.un2.fastq","$unpairedFile");
}
}
my $command = "fastq-join -r $outputDir/mergeLen_dist.txt -p $maxDiffPerc -m $minOverlap $pairedFile -o $outputDir/reads.\%.fastq > $log";
&lprint (" Running \n $command \n");
&executeCommand($command);
&touchFile("$outputDir/runJoinPE.finished");
&printRunTime($time);
if ( -s "$outputDir/reads.join.fastq"){
if($unpairedFile && !$useJoinedPEonly){
&executeCommand("cat $outputDir/reads.join.fastq >> $unpairedFile");
}else{
$unpairedFile = "$outputDir/reads.join.fastq";
}
}
if ($useJoinedPEonly){
return ("","$unpairedFile");
}else{
return ("$outputDir/reads.un1.fastq $outputDir/reads.un2.fastq","$unpairedFile");
}
}
sub runQiimeAnalysis{
my $unpairedFile=shift;
my $pairedFile=shift;
my $time=time();