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runPipeline
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runPipeline
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#!/usr/bin/env perl
use strict;
use File::Basename;
use Getopt::Long;
use LWP::UserAgent;
use Term::ANSIColor;
use POSIX qw(strftime);
use Cwd;
use FindBin qw($RealBin);
use lib "$RealBin/lib";
#use File::Tee qw(tee);
my $workingDir = Cwd::getcwd();
$ENV{PATH} = "$RealBin:$RealBin/bin/:$RealBin/scripts/:$ENV{PATH}";
$ENV{PERL5LIB} = "$RealBin/lib:$ENV{PERL5LIB}";
$ENV{EDGE_HOME} = $RealBin if ! defined $ENV{EDGE_HOME};
$ENV{HOME} ||= $RealBin;
# unbuffer
$|=1;
my $version="1.1";
my $debug=0;
my @unpairedReadsFile;
my @pairedReadsFile;
my $outDir;
my $numCPU=8;
my $configFile;
my @referenceGenome;
my $noColorLog=0;
my $primerFile;
my $proxy=$ENV{HTTP_PROXY};
my ($annoationGFF,$annoationFAA,$annoationFNA); #assembly annotation files
GetOptions("u=s{,}" => \@unpairedReadsFile,
"p=s{,}" => \@pairedReadsFile,
"o=s" => \$outDir,
"ref=s{,}" => \@referenceGenome,
"primer=s" => \$primerFile,
"cpu=i" => \$numCPU,
"config|c=s" => \$configFile,
"noColorLog" => \$noColorLog,
"debug" => \$debug,
"version" => sub{printVersion()},
"help|?" => sub{Usage()} );
sub Usage
{
my $msg=shift;
&lprint ("\nfailed ... ".$msg."\n\n") if $msg;
print <<"END";
Usage: perl $0 [options] -c config.txt -p reads1.fastq reads2.fastq -o out_directory
Version $version
Input File:
-u Unpaired reads, Single end reads in fastq
-p Paired reads in two fastq files and separate by space
-c Config File
Output:
-o Output directory.
Options:
-ref Reference genome file in fasta
It will find the genbank file (same prefix) in the same location if any.
-primer A pair of Primers sequences in strict fasta format
-cpu number of CPUs (default: 8)
-version print verison
END
exit;
}
&Usage("Missing Configuration File.") unless ($configFile);
## Global hash reference for configurations.
my $configuration=&readConfig($configFile);
&Usage("Missing input files.") unless (@unpairedReadsFile or @pairedReadsFile or $configuration->{'DoSRADownload'});
&Usage("Missing output directory path.") unless ($outDir);
`mkdir -p $outDir`;
my $abs_outDir=Cwd::abs_path("$outDir");
# Capture error log
my $start_time_string=&getTmpNameByTime;
my $process_log_file = "$outDir/process.log";
my $error_log_file="$outDir/error.log";
open ( LOG, ">>", $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 (@_);exit 1};
# print whole running command and config file to process log
&lprint("\nProject Start: $start_time_string\n");
print LOG qx/ps -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 $_;}}
close $config_fh;
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);
#&checkDependedPrograms;
#&makeDirectory;
if ($configuration->{'DoSRADownload'})
{
&Download_SRA();
}
@pairedReadsFile = &checkFileAndDir;
my $count_file_list= "$outDir/QcReads/fastqCount.txt";
my ($QCpairFile,$QCunpairFile);
if ($configuration->{DoQC})
{
($QCpairFile,$QCunpairFile)=&runQC(\@unpairedReadsFile, \@pairedReadsFile);
}
else
{
($QCpairFile,$QCunpairFile)=&countFastq(\@unpairedReadsFile, \@pairedReadsFile);
}
if ($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 ($configuration->{DoAssembly})
{
if ($configuration->{assembledContigs}){
($contigFile,$contig_for_annotation_file)=&processProvideContigs();
}else{
if ($configuration->{assembler} =~ /idba/i){
($contigFile,$contig_for_annotation_file)=&runIdbaAssembly($QCpairFile,$QCunpairFile);
}elsif ($configuration->{assembler} =~ /spades/i){
($contigFile,$contig_for_annotation_file)=&runSpadesAssembly($QCpairFile,$QCunpairFile);
}
}
&runReadsToContig($QCpairFile,$QCunpairFile,$contigFile) if ($contigFile);
}
my $tax_kingdom=$configuration->{kingdom};
my $referenceGenome;
my $genbankFile;
my $gff3File;
my $annotateProgram=lc($configuration->{annotateProgram})||"prokka";
($referenceGenome,$genbankFile,$gff3File)=&check_reference_genome(\@referenceGenome) if (@referenceGenome);
if ($referenceGenome)
{
my ($r2gBam,$r2gVCF,$r2gGap)=&runReadsToGenome($QCpairFile,$QCunpairFile,$referenceGenome);
my ($r2gUnmappedPaired,$r2gUnmappedSingle);
# map unmapped reads to refseq
if( $configuration->{MapUnmappedReads} ){
($r2gUnmappedPaired, $r2gUnmappedSingle) = &retrieveUnmappedReads($r2gBam);
&runUnmappedReadsToRefSeq($r2gUnmappedPaired, $r2gUnmappedSingle);
}
# taxonomy analysis
if ($configuration->{DoTaxonomy})
{
if ($configuration->{AllReads})
{
&Reads_taxonomyAssignment($QCpairFile,$QCunpairFile,"$outDir/ReadsBasedAnalysis/Taxonomy");
}
else
{
# retrieve unmapped reads
($r2gUnmappedPaired,$r2gUnmappedSingle) = &retrieveUnmappedReads($r2gBam);
&Reads_taxonomyAssignment($r2gUnmappedPaired,$r2gUnmappedSingle,"$outDir/ReadsBasedAnalysis/UnmappedReads/Taxonomy");
`ln -sf $outDir/ReadsBasedAnalysis/UnmappedReads $outDir/ReferenceBasedAnalysis/`;
}
}
my ($unusedContigFasta,$c2g_SNP_INDEL_file,$c2g_Gap);
if ($configuration->{DoAssembly} and $contigFile)
{
($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);
}
else # reference unknown
{
# Reads taxonomy
&Reads_taxonomyAssignment($QCpairFile,$QCunpairFile,"$outDir/ReadsBasedAnalysis/Taxonomy") if ($configuration->{DoTaxonomy});
#blast nt for contigs taxonomy
&ContigsBlast($contigFile,'nt') if ($configuration->{DoBlast});
}
if ($configuration->{DoContigsTaxonomy} and $contigFile)
{
&Contigs_taxonomyAssignment($contigFile);
}
if ($configuration->{DoAssembly} and $contigFile )
{
#not sure if it is Virus, use size of references to determine the kingdoma (580K is smallest Prok,Mycoplasma)
my $contigTotalSize = `grep contigs.fa $outDir/AssemblyBasedAnalysis/contigs_stats.txt | awk \'{print \$9}\'`;
if (!$tax_kingdom)
{
$tax_kingdom = ($contigTotalSize >= 580000) ? "Bacteria":"Viruses";
}
if ($configuration->{DoAnnotation})
{
($annoationGFF,$annoationFAA,$annoationFNA)=&runAnnotation($contig_for_annotation_file,$tax_kingdom,$annotateProgram,$project_name);
#phagefinder if Bacteria
&phageFinder($annoationGFF,$annoationFAA,$annoationFNA) if ($tax_kingdom ne "Viruses" and $configuration->{DoProPhageDetection});
}
}
if ($configuration->{DoSNPtree})
{
&SNPtree($QCpairFile,$QCunpairFile,$contigFile);
}
my $pcrCheckFailure=0;
if ($primerFile and $configuration->{DoPrimerValidation})
{
$pcrCheckFailure=&pcrValidation($primerFile,$contigFile, $referenceGenome);
}
if ($configuration->{DoAssembly} and $contigFile)
{
#primer Design for Assay Failure and Novel pathgen
if ($pcrCheckFailure == 1 || $configuration->{DoPrimerDesign})
{
&pcrDesign($contigFile);
}
}
# generate JBrowse Tracks
&writeJBrowseInfo($outDir) if ($configuration->{DoJBrowse});
my $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 ($configuration->{DoHTMLReport});
&lprint ("Total");
&printRunTime($^T);
&lprint ("All Done. \n\n");
close LOG;
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 runUnmappedContigToRefSeq
{
my $file=shift;
my $db="$RealBin/database/bwa_index/NCBI-Bacteria-Virus.fna";
my $time=time();
my $outputDir="$abs_outDir/ReferenceBasedAnalysis/UnmappedContigs";
my $prefix="UnmappedContigs";
my $log="$outputDir/log.txt";
if ( -s "$outputDir/UnmappedContigs.ctg_class.csv" )
{
&lprint (" Done mapping $prefix.\n");
return 0;
}
&lprint (" Mapping unmapped contigss to RefSeq...\n");
`mkdir -p $outputDir`;
my $cmd = "$RealBin/scripts/contig_classifier_by_bwa/contig_classifier_by_bwa.pl --db $db --threads $numCPU --prefix $prefix --input $file";
$cmd .= " 1>$log 2>\&1 ";
chdir $outputDir;
&lprint (" Running \n $cmd\n");
&executeCommand($cmd);
chdir $workingDir;
&printRunTime($time);
&lprint (" Done mapping $prefix.\n");
return 0;
}
sub runUnmappedReadsToRefSeq
{
my $pairFile= shift;
my $unpairFile= shift;
my $db="$RealBin/database/bwa_index/NCBI-Bacteria-Virus.fna";
my $time=time();
my $cov_cut_off = 0; #No cutoff filter
my $outPrefix = "UnmappedReads";
my $outputDir= "$outDir/ReferenceBasedAnalysis/UnmappedReads";
my $log= "$outputDir/mapping.log";
if ( -s "$outputDir/UnmappedReads_coverage.txt" )
{
&lprint (" Done mapping $outPrefix.\n");
return 0;
}
`mkdir -p $outputDir`;
&lprint (" Mapping reads that were unmapped to reference(s) to RefSeq...\n");
my $parameters;
$parameters .= " -c $cov_cut_off";
$parameters .= " -p \'$pairFile\'" if ($pairFile);
$parameters .= " -u $unpairFile" if ( -s $unpairFile);
$parameters .= " -bwa_options ". "'" . "-t $numCPU " . $configuration->{"bowtieOptions"}. "'";
$parameters .= " -d $outputDir -pre $outPrefix -ref $db";
my $command = "perl $RealBin/scripts/runReadsToContig.pl $parameters 1>$log 2>\&1 ";
&lprint (" Running \n $command\n");
&executeCommand($command);
$command = "perl $RealBin/scripts/id_mapping_w_gi.pl $outputDir/UnmappedReads_coverage.table reads > $outputDir/UnmappedReads_coverage.txt";
&executeCommand($command);
&lprint (" Done mapping $outPrefix.\n");
&printRunTime($time);
}
sub Contigs_taxonomyAssignment
{
my $file=shift;
my $db="$RealBin/database/bwa_index/NCBI-Bacteria-Virus.fna";
my $time=time();
my $outputDir="$abs_outDir/AssemblyBasedAnalysis/Taxonomy";
my $prefix="$project_name";
my $cov_table="$abs_outDir/AssemblyBasedAnalysis/readsMappingToContig/readsToContigs_coverage.table";
my $tax_assignments_result="$outputDir/$prefix.ctg_class.csv";
my $log="$outputDir/log.txt";
if ($noColorLog)
{
&lprint ("[Contigs Taxonomy Classification]\n");
}
else
{
&lprint (colored ("[Contigs Taxonomy Classification]\n",'yellow'));
}
if ( -e "$outputDir/ContigsTaxonomy.finished" )
{
&lprint ("Contigs Taxonomy Classification Finished\n");
return 0;
}
`mkdir -p $outputDir`;
system("rm -f $outputDir/ContigsTaxonomy.finished");
my $cmd="$RealBin/scripts/contig_classifier_by_bwa/contig_classifier_by_bwa.pl --db $db --threads $numCPU --prefix $prefix --input $file";
$cmd .= " 1>$log 2>&1";
&lprint (" Running \n $cmd\n");
chdir $outputDir;
&executeCommand($cmd);
$cmd="$RealBin/scripts/contig_classification_plot.R $cov_table $tax_assignments_result $prefix";
&lprint (" Running \n $cmd\n");
&executeCommand($cmd);
chdir $workingDir;
&printRunTime($time);
system("touch $outputDir/ContigsTaxonomy.finished");
return 0;
}
sub Download_SRA
{
my $time=time();
my $outputDir="$outDir/SRA_Download";
my $log="$outputDir/log.txt";
if ($noColorLog)
{
&lprint ("[Download SRA]\n");
}
else
{
&lprint (colored ("[Download SRA]\n",'yellow'));
}
if ( -e "$outputDir/DownloadSRA.finished" )
{
&read_SRAD_Download_dir($outputDir);
&lprint ("Download SRA Finished\n");
return 0;
}
`mkdir -p $outputDir`;
system("rm -f $outputDir/DownloadSRA.finished");
my @SRA_ids = split /[:,\s]+/, uc $configuration->{'SRA_id'};
my $cmd = "perl $RealBin/scripts/sra2fastq.pl --gzip --outdir $outputDir --clean ";
$cmd .= join (" ",@SRA_ids);
$cmd .= " 1>$log 2>&1 ";
&lprint (" Running \n $cmd \n");
&executeCommand($cmd);
&read_SRAD_Download_dir($outputDir);
&printRunTime($time);
system("touch $outputDir/DownloadSRA.finished");
return 0;
}
sub read_SRAD_Download_dir
{
my $dir=shift;
opendir(my $sra_dir_fh,$dir) or die "Cannot read $dir\n";
my @files = sort grep {/fastq/} readdir($sra_dir_fh);
if (!@files && !@pairedReadsFile && !@unpairedReadsFile)
{
&lprint("No fastq file from the SRA downloaed. Failed\n");
die "No input\n";
}
if (scalar(@files)==1)
{
push @unpairedReadsFile, "$dir/$files[0]";
}else
{
foreach (@files)
{
if (/_[12]/)
{
push @pairedReadsFile, "$dir/$_";
}else{
push @unpairedReadsFile, "$dir/$_";
}
}
}
close $sra_dir_fh;
}
sub check_reference_genome
{
my $referenceGenome_r = shift;
my $time=time();
my %format;
my $outputDir = "$abs_outDir/Reference/";
my $referenceGenome = "$outputDir/reference.fasta";
my $referenceGBK = "$outputDir/reference.gbk";
my $gff3File="$outputDir/reference.gff";
my $file_list="$outDir/Reference/ref_list.txt";
my @files = @{$referenceGenome_r};
my @fasta_files;
my $check_value=0;
`mkdir -p $outputDir`;
`mkdir -p $outDir/ReferenceBasedAnalysis`;
if (-e $file_list)
{
my %previous_run_ref;
open(my $fh, $file_list) or die "Cannot open $file_list\n";
while(<$fh>)
{
chomp;
$previous_run_ref{$_}=1;
$check_value++;
}
close $fh;
if (scalar(@files) == $check_value)
{
foreach my $file (@files)
{
my ($file_name, $file_path, $file_suffix)=fileparse("$file", qr/\.[^.]*/);
$check_value-- if ($previous_run_ref{$file_name});
}
}
if ($check_value == 0 and -s $referenceGenome) ## same references were used.
{
return($referenceGenome,$referenceGBK,$gff3File);
}
else
{
unlink "$outDir/JBrowse/writeJBrowseInfo.finished";
unlink "$outDir/ReadsBasedAnalysis/readsMappingToRef/runReadsToGenome.finished" ;
unlink "$outDir/ReadsBasedAnalysis/UnmappedReads/Taxonomy/taxonomyAssignment.finished";
unlink "$outDir/AssemblyBasedAnalysis/contigMappingToRef/runContigToGenome.finished";
unlink "$outDir/AssemblyBasedAnalysis/Blast/ContigsBlast.finished";
unlink "$outDir/AssemblyBasedAnalysis/contigMappingToRef/variantAnalysis.finished";
}
}
&lprint (" Checking Reference genome\n");
unlink $referenceGBK;
unlink $gff3File;
unlink $file_list;
foreach my $file (@files)
{
my ($file_name, $file_path, $file_suffix)=fileparse("$file", qr/\.[^.]*/);
&executeCommand("echo $file_name >> $file_list");
if (is_genbank($file))
{
$format{"genbank"}++;
&lprint ("Converting $file_name Genbank to Fasta and GFF\n");
if ($file_suffix =~ /gz/)
{
&executeCommand("gunzip -c $file | genbank2fasta.pl - > $outputDir/$file_name.fasta");
&executeCommand("gunzip -c $file >> $referenceGBK");
}
else
{
&executeCommand("genbank2fasta.pl $file > $outputDir/$file_name.fasta");
&executeCommand("cat $file >> $referenceGBK");
}
push @fasta_files, "$outputDir/$file_name.fasta";
}
if (is_fasta($file))
{
$format{"fasta"}++;
if ($file_suffix =~ /gz/)
{
&executeCommand("gunzip -c $file > $outputDir/$file_name");
$file = "$outputDir/$file_name" ;
}
push @fasta_files, $file;
}
}
&executeCommand("genbank2gff3.pl --outdir stdout $referenceGBK > $gff3File") if ( -s $referenceGBK);
if ( scalar (keys %format) > 1)
{
&lprint("The input refernece mix up with genbank and fasta format. It may cause JBrowse tracks not function properly\n");
}
if (scalar(@fasta_files)>1)
{
my $cmd = "cat ". join(" ",@fasta_files). "> $referenceGenome";
&executeCommand($cmd);
}
else
{
&executeCommand("ln -sf $fasta_files[0] $referenceGenome") if ( ! -s $referenceGenome );
}
my $ref_header = `head -n 1 $referenceGenome`;
if ($ref_header =~ m/virus|viral|phage/i ) {$tax_kingdom= "Viruses";}
my $refSize = &fastaAllSize($referenceGenome);
if ($refSize==0)
{
$referenceGenome="";
&lprint (" The input genome size is 0. Will skip analysis modules require reference genomes\n");
return($referenceGenome,$referenceGBK,$gff3File);
}
#not sure if it is Virus, use size of references to determine the kingdoma (580K is smallest Prok,Mycoplasma)
if (!$tax_kingdom) { $tax_kingdom = ($refSize < 580000)? "Viruses":"Bacteria";}
# replace dot to underscore in the name tag to avoid error on JBrowse
&executeCommand("sed -i.bak 's/[\\|\\.]/_/g' $referenceGenome");
&printRunTime($time);
return($referenceGenome,$referenceGBK,$gff3File);
}
sub phageFinder
{
my $GFF=shift;
my $FAA=shift;
my $FNA=shift;
my $time=time();
my $outputDir="$outDir/AssemblyBasedAnalysis/Prophage";
my $outputDirTmp="$outDir/AssemblyBasedAnalysis/ProphageTmp";
my $outputFile="$outputDir/phageFinder_summary.txt";
my $cmd;
if ($noColorLog)
{
&lprint ("[ProPhage Detection]\n");
}
else
{
&lprint (colored ("[ProPhage Detection]\n",'yellow'));
}
if ( -e "$outputDir/phageFinder.finished")
{
&lprint ("ProPhage Detection Finished\n");
return 0;
}
`mkdir -p $outputDirTmp`;
`mkdir -p $outputDir`;
my $outputDir_abs_path = Cwd::abs_path("$outputDir");
my $outputTmpDir_abs_path = Cwd::abs_path("$outputDirTmp");
if ( -z $FAA || ! -e $FAA)
{
warn "No CDS annotation for phage finder\n";
`rm -rf $outputDirTmp`;
system("touch $outputDir/phageFinder.finished");
&printRunTime($time);
return 0;
}
system("rm -f $outputDir/phageFinder.finished");
unlink "$outDir/JBrowse/writeJBrowseInfo.finished";
# prepare input (Assembly.con,id_map.txt, phage_finder_info.txt) for phage finder.
$cmd="$RealBin/scripts/phageFinder_prepare.pl -o $outputDirTmp $GFF $FNA";
&lprint (" Running \n $cmd\n");
&executeCommand($cmd);
chdir "$outputTmpDir_abs_path";
&executeCommand("ln -sf $FAA Assembly.pep");
#&executeCommand("ln -sf $FNA Assembly.con");
$cmd="$RealBin/thirdParty/phage_finder_v2.1/bin/phage_finder_v2.1.sh Assembly $numCPU 1>$outputDir_abs_path/log.txt 2>\&1";
&lprint (" Running \n $cmd\n");
&executeCommand($cmd);
chdir $workingDir;
# rename the result id back
if ( -e "$outputTmpDir_abs_path/PFPR_tab.txt")
{
my %id_map;
open(my $fh,"$outputTmpDir_abs_path/id_map.txt") or die "Cannot read $outputTmpDir_abs_path/id_map.txt\n";
while(<$fh>){chomp; my($new_id,$original_id)=split; $id_map{$new_id}=$original_id;}
close $fh;
open(my $ofh,">$outputFile") or die "Cannot write $outputFile\n";
open(my $result_fh,"$outputTmpDir_abs_path/PFPR_tab.txt") or die "Cannot read $outputTmpDir_abs_path/PFPR_tab.txt\n";
while(<$result_fh>)
{
my @fields=split /\s+/,$_;
$fields[0]=$id_map{$fields[0]} if ($id_map{$fields[0]});
print $ofh join("\t",@fields),"\n";
}
close $result_fh;
close $ofh;
}
#system("mv $outputTmpDir_abs_path/PFPR_tab.txt $outputFile") if ( -e "$outputTmpDir_abs_path/PFPR_tab.txt");
my $num_phageHit= (-e "$outputFile")? `wc -l $outputFile | awk '{print \$1}'`:1;
&lprint (" Numaber of prophage hit found: ". ($num_phageHit - 1) ."\n");
#print " See $outputFile for the result\n" if (-e "$outputFile");
system("rm -rf $outputDirTmp");
system("touch $outputDir/phageFinder.finished");
&printRunTime($time);
return 0;
}
sub Reads_taxonomyAssignment
{
my $pairFile = shift;
my $unpairFile = shift;
my $outputDir = shift;
my $input = ($outputDir =~ /Unmap/)?"$outputDir/UnmappedReads.fastq":"$outputDir/allReads.fastq";
my $logFile = "$outputDir/error.log";
`mkdir -p $outputDir`;
my $time=time();
my $dparam="--debug" if $debug;
my $bwaScoreCut=30;
#find out the mean of input reads
if ( -e "$outDir/QcReads/QC.stats.txt" ){
open(my $qcfh, "<", "$outDir/QcReads/QC.stats.txt") or die $!;
my $after=0;
foreach(<$qcfh>) {
chomp;
if ($_ =~ /^Mean Reads Length: (.+)/){
$bwaScoreCut = int($1*0.8);
last;
}
}
close ($qcfh);
}
elsif( -e "$outDir/QcReads/fastqCount.txt" ){
open(my $qcfh, "<", "$outDir/QcReads/fastqCount.txt") or die $!;
my ($reads,$bases)=(0,0);
foreach(<$qcfh>) {
chomp;
my @temp = split /\t/, $_;
if(@temp){
$reads += $temp[1];
$bases += $temp[2];
}
}
$bwaScoreCut = int($bases/$reads*0.8);
close ($qcfh);
}
my $cmd0 ="$RealBin/scripts/microbial_profiling/microbial_profiling_configure.pl $RealBin/scripts/microbial_profiling/microbial_profiling.settings.tmpl $configuration->{enabledTools} $bwaScoreCut > $outputDir/microbial_profiling.settings.ini 2>$logFile";
my $cmd="$RealBin/scripts/microbial_profiling/microbial_profiling.pl $dparam -o $outputDir -s $outputDir/microbial_profiling.settings.ini -c $numCPU $input 2>$logFile";
if ($noColorLog)
{
&lprint ("[Reads Taxonomy Classification]\n");
}
else
{
&lprint (colored ("[Reads Taxonomy Classification]\n",'yellow'));
}
if ( -e "$outputDir/taxonomyAssignment.finished")
{
&lprint ("Reads Taxonomy Classification Finished\n");
return 0;
}
system("rm -f $outputDir/taxonomyAssignment.finished");
&executeCommand("cat $pairFile $unpairFile > $input");
&lprint (" Running \n $cmd\n");
&executeCommand($cmd0);
&executeCommand($cmd);
#unlink $input;
$cmd = "$RealBin/scripts/svg2pdf $outputDir/report/*/*/*svg 2>>$logFile";
&executeCommand($cmd) if (system("which inkscape 1>/dev/null") == 0);
system("touch $outputDir/taxonomyAssignment.finished");
&printRunTime($time);
return 0;
}
sub variantAnalysis
{
my $c2g_SNP_INDEL_File=shift;
my $c2g_gap_file=shift;
my $r2g_vcf_file=shift;
my $r2g_gap_file=shift;
my $gff3File=shift;
my $time=time();
my $outputDir="$outDir/AssemblyBasedAnalysis/contigMappingToRef/";
my $log = "$outputDir/variantAnalysis.log";
my $r2g_outputDir="$outDir/ReadsBasedAnalysis/readsMappingToRef/";
#`mkdir -p $outputDir`;
my $c2g_gapAnalysisOutput="$outputDir/GapVSReference.report.txt";
my $r2g_gapAnalysisOutput="$r2g_outputDir/GapVSReference.report.txt";
my $log2 = "$r2g_outputDir/variantAnalysis.log";
my $cmd;
if ($noColorLog)
{
&lprint ("[Variant Analysis]\n");
}
else
{
&lprint (colored ("[Variant Analysis]\n",'yellow'));
}
if ( ! -e "$gff3File")
{
&lprint ("GFF3 file not exists. Skip Variant Analysis\n");
return 0;
}
if ( -e "$outputDir/variantAnalysis.finished")
{
&lprint ("Variant Analysis Finished\n");
return 0;
}
unlink "$outDir/JBrowse/writeJBrowseInfo.finished";
system("rm -f $outputDir/variantAnalysis.finished");
$cmd="$RealBin/scripts/SNP_analysis.pl -gff $gff3File -SNP $c2g_SNP_INDEL_File -format nucmer -output $outputDir 2>$log";
&lprint (" Running \n $cmd\n");
&executeCommand($cmd) if ( -s $c2g_SNP_INDEL_File);
$cmd="$RealBin/scripts/gap_analysis.pl -gff $gff3File -gap $c2g_gap_file > $c2g_gapAnalysisOutput 2>>$log";
&lprint (" Running \n $cmd\n");
&executeCommand($cmd) if ( -s $c2g_gap_file);
$cmd="$RealBin/scripts/SNP_analysis.pl -gff $gff3File -SNP $r2g_vcf_file -format vcf -output $r2g_outputDir 2>>$log2";
&lprint (" Running \n $cmd\n");
&executeCommand($cmd) if ( -s $r2g_vcf_file);
$cmd="$RealBin/scripts/gap_analysis.pl -gff $gff3File -gap $r2g_gap_file > $r2g_gapAnalysisOutput 2>>$log2";
&lprint (" Running \n $cmd\n");
&executeCommand($cmd) if ( -s $r2g_gap_file);
&printRunTime($time);
system("touch $outputDir/variantAnalysis.finished");
return 0;
}
sub ContigsBlast
{
my $file=shift;
my $db=shift;
#my $outputDir=($db eq "nt")?"$outDir/AssemblyBasedAnalysis/Blast":"$outDir/AssemblyBasedAnalysis/contigMappingToRef/";
my $outputDir="$outDir/AssemblyBasedAnalysis/Blast";
`mkdir -p $outputDir`;
my $outputFile="$outputDir/ContigFasta.$db.blast.out";
my $outputFile2="$outputDir/ContigFasta.virulence.blastx";
my $time=time();
system("ln -sf $file $outputDir/ContigsForBlast");
if ($noColorLog)
{
&lprint ("[Contigs Blast] \n to $db and virluence proteins Step ...\n");
}
else
{
&lprint (colored ("[Contigs Blast] \n to $db and virluence proteins ...\n",'yellow'));
}
if ( -e "$outputDir/ContigsBlast.finished" )
{
&lprint ("Contigs Blast Finished\n");
return ($outputFile,$outputFile2);
}
if ( -z $file)
{
&lprint ("Contigs File for Blast is EMPTY\n.");
&printRunTime($time);
system("touch $outputDir/ContigsBlast.finished");
return ($outputFile,$outputFile2);
}
system("rm -f $outputDir/ContigsBlast.finished");
my $blast_program = ($db eq 'nt')? "blastn":"blastx";
my $blastDBPath=$configuration->{"BLAST_${db}_DB"};
# Need to modified the path
my $cmd = "$blast_program -db $blastDBPath/$db -query $file -out $outputFile -num_threads $numCPU -evalue 1e-10 -outfmt '6 std staxids' ";
if ( -f "$blastDBPath/nt.00.nsq" || -f "$blastDBPath/nr.00.psq")
{
&lprint (" Running \n $cmd\n");
&executeCommand($cmd);
$cmd="ktImportBLAST -o $outputFile.kronaInternet.html $outputFile:$outDir/AssemblyBasedAnalysis/readsMappingToContig/magnitudes.txt ";
&lprint (" Running \n $cmd\n");
&executeCommand($cmd);
$cmd="$RealBin/scripts/krona_portable.pl --inhtml $outputFile.kronaInternet.html --outhtml $outputFile.krona.html";
&executeCommand($cmd);
unlink "$outputFile.kronaInternet.html";
$cmd="$RealBin/scripts/ClassifyBLAST.pl -o $outputFile.classification $outputFile";
&lprint (" Running \n $cmd\n");
&executeCommand($cmd);
}
else
{
&lprint ("Cannot find BLAST $db database, step SKIP ...\n");
}
my $faa_bytes = -s $file;
my $bsize = int($faa_bytes / $numCPU / 2);
my $blastxcmd= "blastx -db $RealBin/database/MvirDB/virulenceDB.protein.fasta -query - -evalue 1e-10 -outfmt '6 std stitle qcovs qcovhsp'";
$cmd = "cat $file | parallel -j$numCPU --block $bsize --recstart '>' --pipe \"$blastxcmd\" > $outputFile2 2> /dev/null";
&lprint (" Running \n $cmd\n");
&executeCommand($cmd);
&printRunTime($time);
system("touch $outputDir/ContigsBlast.finished");
return ($outputFile,$outputFile2);
}
sub retrieveUnmappedReads
{
my $bamFile=shift;
my $time=time();
my $outputDir="$outDir/ReadsBasedAnalysis/UnmappedReads";
my $unmappedSingle="$outputDir/singleEnd.fastq";
my $unmappedPaired_1="$outputDir/pairedEnd.1.fastq";
my $unmappedPaired_2="$outputDir/pairedEnd.2.fastq";
my $NumOfunmappedPaired=0;
my $NumOfunmappedSingle=0;
`mkdir -p $outputDir`;
if ( (-s "$unmappedSingle" || -s "$unmappedPaired_1") && -e "$outputDir/retrieveUnmappedReads.finished" )
{
&lprint (" Unmapped reads retrieved\n");
if ($QCpairFile)
{
return ("$unmappedPaired_1 $unmappedPaired_2",$unmappedSingle);
}
else
{
return ("",$unmappedSingle);
}
}
else
{
&lprint (" Retrieveing unmapped reads", "\n");
system("rm -f $outputDir/retrieveUnmappedReads.finished");
}
open (my $se_fh,"> $unmappedSingle") or die "cannot open $unmappedSingle to write\n";
my ($pe_fh1, $pe_fh2);
if ($QCpairFile)
{
open ($pe_fh1,">$unmappedPaired_1") or die "cannot open $unmappedPaired_1 to write\n";
open ($pe_fh2,">$unmappedPaired_2") or die "cannot open $unmappedPaired_2 to write\n";
&executeCommand("samtools view -f 4 $bamFile | sort -T $outputDir -k 1 > $outputDir/unmapped.sam");
}
else
{
&executeCommand("samtools view -f 4 $bamFile > $outputDir/unmapped.sam");
}
open (my $fh, "$outputDir/unmapped.sam") or die "cannot open $outputDir/unmapped.sam\n";
while (<$fh>)
{
chomp;
my @samFields=split /\t/,$_;
if ($samFields[1] & 1) # paired reads
{
if (($samFields[1] & 4) && ($samFields[1] & 8)) # both unmapped
{
$NumOfunmappedPaired = $NumOfunmappedPaired + 1 ;
print $pe_fh1 "@".$samFields[0]."/1\n".$samFields[9]."\n+\n".$samFields[10]."\n" if ($samFields[1] & 64);
print $pe_fh2 "@".$samFields[0]."/2\n".$samFields[9]."\n+\n".$samFields[10]."\n" if ($samFields[1] & 128);
}
else
{
# the other mate mapped, do we need to keep this?
$NumOfunmappedSingle++;
print $se_fh "@".$samFields[0]."\n".$samFields[9]."\n+\n".$samFields[10]."\n";
}
}
else # single end reads
{
# original from single end reads and unmapped. Could be after QC SE reads.
$NumOfunmappedSingle++;
print $se_fh "@".$samFields[0]."/1\n".$samFields[9]."\n+\n".$samFields[10]."\n";
}
}
close $fh;
close $se_fh;
&lprint (" Unmapped reads:\n");
&lprint (" Paired End: $NumOfunmappedPaired\n");
&lprint (" Single End: $NumOfunmappedSingle\n");
unlink "$outputDir/unmapped.sam";
&printRunTime($time);
system("touch $outputDir/retrieveUnmappedReads.finished");
if ($QCpairFile)
{
return ("$unmappedPaired_1 $unmappedPaired_2",$unmappedSingle);
}
else
{
return ("",$unmappedSingle);
}
}
sub runAnnotation
{
my $contigFile= shift;
my $tax_kingdom = shift;
my $annotateProgram=shift;
my $project_name=shift;
my $time=time();
my $outputDir= "$outDir/AssemblyBasedAnalysis/Annotation";
`mkdir -p $outputDir`;
my $outputDir_abs_path = Cwd::abs_path("$outputDir");
my $outGenbank="$outputDir_abs_path/$project_name.gbk";
my $outGFF="$outputDir_abs_path/$project_name.gff";
my $outFAA="$outputDir_abs_path/$project_name.faa";
my $outFNA="$outputDir_abs_path/$project_name.fna";
my $log = "$outputDir/Annotation.log";
my $gff_plot_log = "$outputDir/plot_gff3.log";
my $cmd;
if ($noColorLog)
{
&lprint ("[Contigs Annotation]\n");
}
else
{
&lprint (colored ("[Contigs Annotation]\n",'yellow'));
}
#if ( -e "$log")
if ( -e "$outputDir/runAnnotation.finished")
{
#if (`grep "Thank you" $log` || `grep "Share and enjoy" $log`)
#{
&lprint ("Contig Annotation Finished\n");
return ($outGFF,$outFAA,$outFNA);
#}
}
unlink "$outDir/JBrowse/writeJBrowseInfo.finished";
if ( -z $contigFile || ! -e $contigFile){
&lprint ("failed: No contigs > $contig_size_for_annotation bp for annotation\n");
`echo "No contigs > $contig_size_for_annotation bp for annotation" >$log`;
&printRunTime($time);
system("touch $outputDir/runAnnotation.finished");
return ($outGFF,$outFAA,$outFNA);
}
if ($annotateProgram =~ /RATT/i)
{
my $sourceGBK = $configuration->{annotateSourceGBK};
if (!$sourceGBK || ! is_genbank($sourceGBK)) {
&lprint("No source reference GenBank file for RATT annotation transfer. Will use prokka to do annotation\n");
$annotateProgram = "prokka";
}else{
`mkdir -p $outputDir_abs_path/RATT/source`;
chdir "$outputDir_abs_path/RATT";
my $ref_abs_path = "$outputDir_abs_path/RATT/source";
my $contig_abs_path = Cwd::abs_path("$contigFile");
# convert genbank to embl for RATT annoation transfer
&executeCommand("cp $sourceGBK $outputDir_abs_path/RATT/source/source.gbk");
&executeCommand("genbank2embl.pl $outputDir_abs_path/RATT/source/source.gbk 2>/dev/null");
$cmd = ("$RealBin/scripts/runRATT.sh $ref_abs_path $contig_abs_path $project_name Species 1>$log 2>\&1 ");
}
}