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epiTEome.pl
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#!/usr/bin/perl
use strict;
use Getopt::Long ;
use warnings ;
use File::Basename ;
use Bio::SeqIO ;
use Bio::Seq::Quality;
use Bio::Tools::GFF;
use Bio::DB::Sam;
use Bio::DB::Bam::Alignment;
use Cwd;
use File::Which;
use Set::IntervalTree ;
use Statistics::Descriptive;
# use Devel::Size qw(total_size); # Need to be remove for public version
use Data::Dumper ;
my $prog = basename($0) ;
my $VERSION = '1.0';
my $lastmodif = '2017-02-01';
my $help;
my ($gffFile, $unmappedFastqFile, $targetList, $refFasta) ;
my $threads = 1 ;
my $bamFile ;
my $batchNb = 5000 ;
my $samOutputFile ;
my $lengthPairedEnd = '25,30,40' ;
my $windowSize = 10 ;
my $displayMet = "metaplot" ;
my $debug ;
my $noclean ;
my $segemehl_Eval = 5 ;
my $segemehl_D = 1 ;
&GetOptions(
"gff:s" => \$gffFile,
"un:s" => \$unmappedFastqFile,
"t:s" => \$targetList,
"ref:s" => \$refFasta,
"noclean" => \$noclean,
"p:i" => \$threads,
"b:i" => \$batchNb,
"chop:s" => \$lengthPairedEnd,
"w:i" => \$windowSize,
"metO:s" => \$displayMet,
"debug" => \$debug,
"E:i" => \$segemehl_Eval,
"D:i" => \$segemehl_D,
"h|help" => \$help);
$help and &help;
&main();
sub main {
my $self = {};
bless $self;
my $datestring = localtime();
print STDERR "INFO $prog $datestring Start program!\n" ;
$self->setOptions();
$self->checkPrograms();
$self->readTargetFile();
$self->sF_TSDsize();
$self->readGffFile();
#---- Uncompress INPUT FASTQ if necessary
my @unCompress ;
if($self->{option}->{unmappedFastqFile}->{file}=~/(.*).bz2$/){
@unCompress = ($self->{program}->{bzip2}, '-d', $self->{option}->{unmappedFastqFile}->{file}) ;
$self->{option}->{unmappedFastqFile}->{file} = $1 ;
}
elsif($self->{option}->{unmappedFastqFile}->{file}=~/(.*).gz$/){
@unCompress = ($self->{program}->{gzip}, '-d', $self->{option}->{unmappedFastqFile}->{file}) ;
$self->{option}->{unmappedFastqFile}->{file} = $1 ;
}
# print STDERR join ( " ", @unCompress),"\n" ;
system(join" ", @unCompress) ;
#---- END
#---- STEP 1: mapping of 'paired-end'
$datestring = localtime();
print STDERR "\nINFO $prog $datestring STEP 1: read ends mapping.\n" ;
$self->{fileName}->{mappingOutput}->{bam} = $self->{option}->{unmappedFastqFile}->{prefix}.".step1.sort.bam" ;
if(-e $self->{fileName}->{mappingOutput}->{bam}){
print STDERR "INFO $prog: --> Skiping Step 1: $self->{fileName}->{mappingOutput}->{bam} allready exist.\n" ;
$self->{fileName}->{splitFastq}->{inFastq} = $self->{option}->{unmappedFastqFile}->{file} ;
$self->splitFastq("PaireEnd", "subset"); # splitFastq sub to find reads length of fastq file.
}
else{
$self->{fileName}->{splitFastq}->{inFastq} = $self->{option}->{unmappedFastqFile}->{file} ;
$self->splitFastq("PaireEnd");
$self->{fileName}->{segemehl}->{-i} = $self->{fileName}->{refFasta}->{file}.'.ctidx' ;
$self->{fileName}->{segemehl}->{-j} = $self->{fileName}->{refFasta}->{file}.'.gaidx' ;
$self->{fileName}->{segemehl}->{-d} = $self->{fileName}->{refFasta}->{file} ;
$self->{fileName}->{segemehl}->{-q} = $self->{fileName}->{splitFastq}->{outFastq} ;
$self->{fileName}->{segemehl}->{-o} = $self->{option}->{unmappedFastqFile}->{prefix}.".step1.sam" ;
$self->{fileName}->{segemehl}->{-Eval} = $segemehl_Eval ;
$self->{fileName}->{mappingOutput}->{bam} = $self->{option}->{unmappedFastqFile}->{prefix}.".step1.sort.bam" ;
$self->mappingMethylCSeq();
if(-z $self->{option}->{unmappedFastqFile}->{prefix}.'.step1.sort.bam'){
print STDERR "DIE $prog: File $self->{option}->{unmappedFastqFile}->{prefix}.'.step1.sort.bam' is empty... die!\n" and die ;
}
my @rm = ("rm", $self->{fileName}->{splitFastq}->{outFastq});
system(join" ", @rm) ;
}
#---- END STEP 1
#---- STEP 2: mapping split-reads
$datestring = localtime();
print STDERR "\nINFO $prog $datestring STEP 2: split-reads mapping.\n" ;
for(1..$self->{batch}){
$datestring = localtime();
print STDERR "\nINFO $prog $datestring BATCH #$_.\n" ;
$self->{fileName}->{mappingOutput}->{bam} = $self->{option}->{unmappedFastqFile}->{prefix}.".step1.sort.bam" ;
foreach my $feat(sort{$self->{targetFeatTmp}->{$a}->{batch} <=> $self->{targetFeatTmp}->{$a}->{batch}} keys %{$self->{targetFeatTmp}}){
if($self->{targetFeatTmp}->{$feat}->{batch} == $_){
$self->{targetFeat}->{$feat} = $self->{targetFeatTmp}->{$feat} ;
}
}
$self->step2() ;
$self->{cleanNum} = 1 ;
if(not defined $noclean){
$self->clean($self->{cleanNum}) ;
}
delete($self->{targetFeat}) ;
}
#---- END STEP 2
$self->makeWindows();
$self->print() ;
$self->printBam();
$self->printRowDataMet();
$self->calcMetPercentage();
$self->calcMeanAndCi();
$self->printMetaplot();
$self->{cleanNum} = 2 ;
if(not defined $noclean){
$self->clean($self->{cleanNum}) ;
}
# compress starting fastq input
my @bzip2 = ($self->{program}->{bzip2}, $self->{option}->{unmappedFastqFile}->{file} ) ;
print STDERR join ( " ", @bzip2),"\n" ;
system(join" ", @bzip2) ;
$datestring = localtime();
print STDERR "INFO $prog $datestring Finishied succesfully !\n" ;
exit(0);
} # END OF main
###########
### sub ###
###########
sub setOptions {
my $self = shift;
$self->{dirName}->{currentDir} = cwd ;
$self->{option}->{maxReadsLength} = 0 ;
if (defined ($gffFile)){
unless (-e $gffFile){ print STDERR "DIE $prog: Could not find file: " . $gffFile . "\n" and die ; }
$self->{option}->{gff} = $gffFile ;
}
else{
print STDERR "ERROR $prog: Need to specify option -gff.\n" and &help ;
}
if (defined ($unmappedFastqFile)){
unless (-e $unmappedFastqFile){ print STDERR "DIE $prog: Could not find file: " . $unmappedFastqFile . "\n" and die ; }
$self->{dirName}->{unDir} = dirname($unmappedFastqFile) ;
$self->{option}->{unmappedFastqFile}->{file} = $unmappedFastqFile ;
if($unmappedFastqFile =~/(.*).fastq$/){
$self->{option}->{unmappedFastqFile}->{prefix} = $1 ;
}
elsif($unmappedFastqFile =~/(.*).fq$/){
$self->{option}->{unmappedFastqFile}->{prefix} = $1 ;
}
elsif($unmappedFastqFile =~/(.*).fastq.bz2$/){
$self->{option}->{unmappedFastqFile}->{prefix} = $1 ;
}
elsif($unmappedFastqFile =~/(.*).fq.bz2$/){
$self->{option}->{unmappedFastqFile}->{prefix} = $1 ;
}
elsif($unmappedFastqFile =~/(.*).fastq.gz$/){
$self->{option}->{unmappedFastqFile}->{prefix} = $1 ;
}
elsif($unmappedFastqFile =~/(.*).fq.gz$/){
$self->{option}->{unmappedFastqFile}->{prefix} = $1 ;
}
else{
print STDERR "ERROR $prog: File give in -un option should have extention fastq or fq.\n" and &help ;
}
}
else{
print STDERR "ERROR $prog: Need to specify option -un.\n" and &help ;
}
if (defined ($targetList)){
unless (-e $targetList){ print STDERR "DIE $prog: Could not find file: " . $targetList . "\n" and die ; }
$self->{option}->{targetList} = $targetList ;
}
else{
print STDERR "ERROR $prog: Need to specify option -t.\n" and &help ;
}
if (defined ($targetList)){
unless (-e $targetList){ print STDERR "DIE $prog: Could not find file: " . $targetList . "\n" and die ; }
$self->{option}->{targetList} = $targetList ;
}
else{
print STDERR "ERROR $prog: Need to specify option -t.\n" and &help ;
}
if (defined ($refFasta)){
unless (-e $refFasta){ print STDERR "DIE $prog: Could not find file: " . $refFasta . "\n" and die ; }
if($refFasta =~/(.*).fasta/ or $refFasta =~/(.*).fna/){
unless (-e $refFasta.".ctidx"){ print STDERR "DIE $prog: Could not find segemehl index file .ctidx \n" and &help ; }
unless (-e $refFasta.".gaidx"){ print STDERR "DIE $prog: Could not find segemehl index file .gaidx \n" and &help ; }
$self->{fileName}->{refFasta}->{file} = $refFasta ;
}
else{
print STDERR "ERROR $prog: File give in -fasta option should have extention fasta or fna.\n" and &help ;
}
}
else{
print STDERR "ERROR $prog: Need to specify option -fasta.\n" and &help ;
}
if(defined $lengthPairedEnd){
if($lengthPairedEnd =~/,/){
my @array = split(",", $lengthPairedEnd) ;
foreach my $nb (sort @array){
push(@{$self->{option}->{lengthPairedEnd}}, $nb) ;
}
}
else{
push(@{$self->{option}->{lengthPairedEnd}}, $lengthPairedEnd) ;
}
}
}
sub checkPrograms{
my $self = shift;
$self->{program}->{bzip2} = which 'bzip2';
if(not defined $self->{program}->{bzip2}){ print STDERR "DIE $prog: Missing program: bzip2.\n" and die ; }
$self->{program}->{gzip} = which 'gzip';
if(not defined $self->{program}->{gzip}){ print STDERR "DIE $prog: Missing program: gzip.\n" and die ; }
$self->{program}->{samtools} = which 'samtools';
if(not defined $self->{program}->{samtools}){ print STDERR "DIE $prog: Missing program: samtools.\n" and die ; }
$self->{program}->{maskFastaFromBed} = which 'maskFastaFromBed';
if(not defined $self->{program}->{maskFastaFromBed}){ print STDERR "DIE $prog: Missing program: maskFastaFromBed.\n" and die ; }
$self->{program}->{fastqutils} = which 'fastqutils';
if(not defined $self->{program}->{fastqutils}){ print STDERR "DIE $prog: Missing program: fastqutils.\n" and die ; }
$self->{program}->{bamutils} = which 'bamutils';
if(not defined $self->{program}->{bamutils}){ print STDERR "DIE $prog: Missing program: bamutils.\n" and die ; }
$self->{program}->{segemehl} = which 'segemehl.x';
if(not defined $self->{program}->{segemehl}){ print STDERR "DIE $prog: Missing program: segemehl.x.\n" and die ; }
$self->{program}->{rm} = which 'rm';
if(not defined $self->{program}->{rm}){ print STDERR "DIE $prog: Missing program: rm.\n" and die ; }
$self->{program}->{awk} = which 'awk';
if(not defined $self->{program}->{awk}){ print STDERR "DIE $prog: Missing program: awk.\n" and die ; }
# print STDERR "INFO $prog: All programs have been found succesfully.\n" ;
}
sub step2{
my $self = shift;
######################
#### If only for development
if(-e $self->{option}->{unmappedFastqFile}->{prefix}.".step2.sort.bam"){ # Only for developpement
print STDERR "INFO $prog: --> Skiping Step 2: $self->{option}->{unmappedFastqFile}->{prefix}.step2.sort.bam allready exist.\n" ;
$self->{fileName}->{mappingOutput}->{bam} = $self->{option}->{unmappedFastqFile}->{prefix}.".step2.sort.bam" ;
#check if $self->{fileName}->{mappingOutput}->{bam} isn't either empty or troncated
if(-z $self->{fileName}->{mappingOutput}->{bam}){
print STDERR "INFO $prog: $self->{fileName}->{mappingOutput}->{bam} is empty.\n" ;
return (0) ;
}
my $out=`samtools view -H $self->{fileName}->{mappingOutput}->{bam} | egrep -c "\@SQ"`;
chomp $out ;
if($out == 0){
print STDERR "INFO $prog: $self->{fileName}->{mappingOutput}->{bam} header troncated.\n" ;
return (0) ;
}
$self->selectSplitReads_v2(); # select split-reads mapped on the targeted TEs.
$self->splitReadsGrab_v2();
$self->groupOverlapingmNeoReads();
$self->neoInsertionFinder();
}
else{
$self->selectPairedEndReads();
$self->pairedEndGrab();
$self->checkPairedEndPosition_v3(); # regarding other copies in the same family
my @fastqutils = ($self->{program}->{fastqutils} ,'filter -whitelist', $self->{option}->{unmappedFastqFile}->{prefix}.'.step1.list', $self->{option}->{unmappedFastqFile}->{file}, ' > ', $self->{option}->{unmappedFastqFile}->{prefix}.'.step1.fastq') ;
print STDERR join ( " ", @fastqutils),"\n" ;
system(join" ", @fastqutils) ;
if(-z $self->{option}->{unmappedFastqFile}->{prefix}.'.step1.fastq'){
print STDERR "INFO $prog: $self->{option}->{unmappedFastqFile}->{prefix}.'.step1.fastq' is empty.\n" ;
return (0) ;
}
# step 2 mapping
$self->{fileName}->{mappingOutput}->{bam} = $self->{option}->{unmappedFastqFile}->{prefix}.".step2.sort.bam" ;
$self->{fileName}->{splitFastq}->{inFastq} = $self->{option}->{unmappedFastqFile}->{prefix}.'.step1.fastq' ;
$self->splitFastq("eachNucl");
$self->{fileName}->{segemehl}->{-i} = $self->{fileName}->{refFasta}->{file}.'.ctidx' ;
$self->{fileName}->{segemehl}->{-j} = $self->{fileName}->{refFasta}->{file}.'.gaidx' ;
$self->{fileName}->{segemehl}->{-d} = $self->{fileName}->{refFasta}->{file} ;
$self->{fileName}->{segemehl}->{-q} = $self->{fileName}->{splitFastq}->{outFastq} ;
$self->{fileName}->{segemehl}->{-o} = $self->{option}->{unmappedFastqFile}->{prefix}.".step2.sam" ;
$self->{fileName}->{mappingOutput}->{bam} = $self->{option}->{unmappedFastqFile}->{prefix}.".step2.sort.bam" ;
$self->{fileName}->{segemehl}->{-Eval} = $segemehl_Eval ;
$self->mappingMethylCSeq();
#check if $self->{fileName}->{mappingOutput}->{bam} isn't either empty or troncated
if(-z $self->{option}->{unmappedFastqFile}->{prefix}.".step2.sort.bam"){
print STDERR "INFO $prog: $self->{fileName}->{mappingOutput}->{bam} is empty.\n" ;
return (0) ;
}
my $out=`samtools view -H $self->{fileName}->{mappingOutput}->{bam} | egrep -c "\@SQ"`;
chomp $out ;
if($out == 0){
print STDERR "INFO $prog: $self->{fileName}->{mappingOutput}->{bam} header troncated.\n" ;
return (0) ;
}
$self->selectSplitReads_v2(); # select split-reads mapped on the targeted TEs.
$self->splitReadsGrab_v2();
$self->groupOverlapingmNeoReads();
$self->neoInsertionFinder();
}
}
sub clean {
my $self = shift ;
my $num = shift ;
my $file ;
$file->{$self->{option}->{unmappedFastqFile}->{prefix}.".pe.fastq"} = 1 ;
$file->{$self->{option}->{unmappedFastqFile}->{prefix}.".matePairedEnd.bam"} = 1 ;
$file->{$self->{option}->{unmappedFastqFile}->{prefix}.".matePairedEnd.bam.bai"} = 1 ;
$file->{$self->{option}->{unmappedFastqFile}->{prefix}.".fishReads.list"} = 1 ;
$file->{$self->{option}->{unmappedFastqFile}->{prefix}.".step1.list"} = 1 ;
$file->{$self->{option}->{unmappedFastqFile}->{prefix}.'.step1.fastq'} = 1 ;
$file->{$self->{option}->{unmappedFastqFile}->{prefix}.".step1.sr.fastq"} = 1 ;
$file->{$self->{option}->{unmappedFastqFile}->{prefix}.".step2.sort.bam"} = 1 ;
$file->{$self->{option}->{unmappedFastqFile}->{prefix}.".step2.sort.bam.bai"} = 1 ;
$file->{$self->{option}->{unmappedFastqFile}->{prefix}.".mateSplitReads.bam"} = 1 ;
$file->{$self->{option}->{unmappedFastqFile}->{prefix}.".mateSplitReads.bam.bai"} = 1 ;
$file->{$self->{option}->{unmappedFastqFile}->{prefix}.".step1.sort.bam"} = 2 ;
$file->{$self->{option}->{unmappedFastqFile}->{prefix}.".step1.sort.bam.bai"} = 2 ;
$file->{"log"} = 2 ;
# $file->{$self->{fileName}->{refSeqNewPrefix}."_refMasked*"} = 1 ;
my @rm = $self->{program}->{rm} ;
foreach my $fileName(sort keys %{$file}){
if($file->{$fileName} <= $num){
if(-e $fileName){
push(@rm, $fileName) ;
}
}
}
if(scalar @rm>1){
print STDERR "INFO $prog: Cleaning file: " ;
print STDERR join ( " ", @rm),"\n" ;
system(join" ", @rm) ;
}
}
sub readTargetFile {
my $self = shift;
open("T",$self->{option}->{targetList}) or print STDERR "Die: Could not open file: " . $self->{option}->{targetList} . "\n" and exit ;
while(<T>){
chomp $_ ;
$self->{targetList}->{$_} = 1 ;
}
}
sub readGffFile {
my $datestring = localtime();
print STDERR "INFO $prog $datestring Run Module: readGffFile!\n" ;
my $self = shift;
my $gffio = Bio::Tools::GFF->new(-file => $self->{option}->{gff}, -gff_version => 3);
my $feature;
my $checkConcodanceGffTarget = "FALSE" ;
my $nbTargetTEsPerFam ;
# loop over the input stream
while($feature = $gffio->next_feature()) {
# if ( $feature->strand eq "+" ){$feature->strand = 1 ; }
# if ( $feature->strand eq "-" ){$feature->strand = -1 ;}
if($feature->has_tag('ID') and $feature->has_tag('fam')){
my @id = $feature->get_tag_values('ID') ;
my @fam = $feature->get_tag_values('fam') ;
my $family = $fam[0] ;
if($fam[0]=~/ATHILA.*/){
$family = "ATHILA" ;
$feature->remove_tag('fam');
$feature->add_tag_value('fam',"ATHILA");
}
$self->{family}->{$family}->{$id[0]} = $feature ;
# Index region into interval tree
my $obj = {
name => $id[0],
start => $feature->start-50,
stop => $feature->end+50,
strand => $feature->strand
};
$self->{bedTree}->{$family}->{$feature->seq_id} = Set::IntervalTree->new unless ( defined $self->{bedTree}->{$family}->{$feature->seq_id} );
$self->{bedTree}->{$family}->{$feature->seq_id}->insert( $obj, $feature->start-50, $feature->end+50);
if(defined $self->{targetList}->{$id[0]}){
if($feature->end-$feature->start+1>$self->{option}->{maxReadsLength}){
$checkConcodanceGffTarget = "TRUE" ;
$self->{targetFeatTmp}->{$id[0]}->{$feature->primary_tag} = $feature ;
$nbTargetTEsPerFam->{$family} ++ ;
}
}
}
elsif($feature->has_tag('Parent') and ($feature->primary_tag eq "LTR5" ) or ($feature->primary_tag eq "LTR3") ){
my @parent = $feature->get_tag_values('Parent') ;
if(defined $self->{targetList}->{$parent[0]}){
if($feature->end-$feature->start+1>$self->{option}->{maxReadsLength}){
$self->{targetFeatTmp}->{$parent[0]}->{$feature->primary_tag} = $feature ;
}
}
}
}
$gffio->close();
# deal with batch
$self->{batch} = 1 ;
my $nbCopiesWithinFam = 0 ;
foreach my $family (sort keys %{$nbTargetTEsPerFam}){
$nbCopiesWithinFam += $nbTargetTEsPerFam->{$family} ;
if($nbCopiesWithinFam >= $batchNb){
$nbCopiesWithinFam = 0 ;
$self->{batch}++ ;
}
foreach my $teid (sort keys %{$self->{family}->{$family}}){
if (defined $self->{targetFeatTmp}->{$teid}){
$self->{targetFeatTmp}->{$teid}->{batch} = $self->{batch} ;
}
}
}
if($checkConcodanceGffTarget eq "FALSE"){
print STDERR "DIE $prog: Features ID in target file (-t) are not present in gff file. Die.\n" and die ;
}
delete($self->{targetList}) ;
# $self->{targetList} = () ;
# delete($nbTargetTEsPerFam) ;
return(1) ;
}
sub splitFastq {
my $datestring = localtime();
print STDERR "INFO $prog $datestring Run Module: splitFastq!\n" ;
my $self = shift ;
my $method = shift ;
my $subset = shift ;
my $k = 1 ;
my $step = 1 ;
my $min = $self->{option}->{lengthPairedEnd}[0] ;
my $prefix ;
unless (-e $self->{fileName}->{splitFastq}->{inFastq}){ print STDERR "DIE $prog: Could not find file: " . $self->{fileName}->{splitFastq}->{inFastq} . "\n" and die ; }
if($self->{fileName}->{splitFastq}->{inFastq} =~/(.*).fastq$/){
$prefix = $1 ;
}
elsif($self->{fileName}->{splitFastq}->{inFastq} =~/(.*).fq$/){
$prefix = $1 ;
}
elsif($self->{fileName}->{splitFastq}->{inFastq} =~/(.*).fastq.bz2$/){
$prefix = $1 ;
}
elsif($self->{fileName}->{splitFastq}->{inFastq} =~/(.*).fq.bz2$/){
$prefix = $1 ;
}
else{
die and print STDERR "DIE $prog: Only .fastq or .fq exention accepted in $self->{fileName}->{splitFastq}->{inFastq}\n" ;
}
my $r1 ;
my $fileName ;
if($method eq "eachNucl"){
$fileName = $prefix.".sr.fastq" ;
$self->{fileName}->{splitFastq}->{outFastq} = $prefix.".sr.fastq" ;
$self->{fileName}->{splitFastq}->{prefix} = $prefix ;
}
elsif($method eq "PaireEnd"){
$fileName = $prefix.".pe.fastq" ;
$self->{fileName}->{splitFastq}->{outFastq} = $prefix.".pe.fastq" ;
$self->{fileName}->{splitFastq}->{prefix} = $prefix ;
}
open($r1, '>', $fileName) or die("Could not open file.\n");
my $in = Bio::SeqIO->new(-format => 'fastq',
-file => $self->{fileName}->{splitFastq}->{inFastq});
while (my $data = $in->next_dataset) {
if(defined $subset){
$k++ ;
if($k>1000){ last ;}
}
my $seq =$data->{"-seq"} ;
my $length = length($seq) ;
if($length > $self->{option}->{maxReadsLength}){$self->{option}->{maxReadsLength} = $length ;}
if($method eq "eachNucl"){
if(defined $self->{storeChop}->{$data->{"-id"}}){ # if you delete checkPairedEndPosition_v3, you should delete also this part.
$min = $self->{storeChop}->{$data->{"-id"}} ;
}
for (my $i = $min ; $i<$length-$min ; $i = $i+$step){
my $id = $data->{'-id'}."_".$i."_".$length ;
print $r1 "@".$id."/1\n" ;
print $r1 (substr $data->{"-seq"}, 1, $i) ;
print $r1 "\n" ;
print $r1 "+\n" ;
print $r1 (substr $data->{"-raw_quality"}, 1, $i) ;
print $r1 "\n" ;
# h2
print $r1 "@".$id."/2\n" ;
print $r1 (substr $data->{"-seq"}, $i+1, $length);
print $r1 "\n" ;
print $r1 "+\n" ;
print $r1 (substr $data->{"-raw_quality"}, $i+1, $length) ;
print $r1 "\n" ;
}
}
elsif($method eq "PaireEnd"){
foreach my $lengthPairedEnd (@{$self->{option}->{lengthPairedEnd}}){
my $id = $data->{'-id'} ;
# h1
print $r1 "@".$id."_".$lengthPairedEnd."/1\n" ;
print $r1 (substr $data->{"-seq"}, 1, $lengthPairedEnd) ;
print $r1 "\n" ;
print $r1 "+\n" ;
print $r1 (substr $data->{"-raw_quality"}, 1, $lengthPairedEnd) ;
print $r1 "\n" ;
# h2
print $r1 "@".$id."_".$lengthPairedEnd."/2\n" ;
print $r1 (substr $data->{"-seq"}, ($length-$lengthPairedEnd), $length);
print $r1 "\n" ;
print $r1 "+\n" ;
print $r1 (substr $data->{"-raw_quality"}, ($length-$lengthPairedEnd), $length) ;
print $r1 "\n" ;
}
}
}
close $r1 ;
if($method eq "eachNucl"){ delete($self->{storeChop}) ; }
}
sub mappingMethylCSeq {
my $datestring = localtime();
print STDERR "INFO $prog $datestring Run Module: Segemehl mapping !\n" ;
my $self = shift ;
# check presence of file
unless(-e $self->{fileName}->{segemehl}->{-i}){
print STDERR "DIE $prog: Could not file Segemehl input file: $self->{fileName}->{segemehl}->{-i}.\n" and &help ;
}
unless(-e $self->{fileName}->{segemehl}->{-d}){
print STDERR "DIE $prog: Could not file Segemehl input file: $self->{fileName}->{segemehl}->{-d}.\n" and &help ;
}
unless(-e $self->{fileName}->{segemehl}->{-q}){
print STDERR "DIE $prog: Could not file Segemehl input file: $self->{fileName}->{segemehl}->{-q}.\n" and &help ;
}
my @map = ($self->{program}->{segemehl}, '--silent -t ',$threads, '-D', $segemehl_D, '-E ', $self->{fileName}->{segemehl}->{-Eval}, ' -i ', $self->{fileName}->{segemehl}->{-i}, '-j', $self->{fileName}->{segemehl}->{-j} , '-d', $self->{fileName}->{segemehl}->{-d}, '-q', $self->{fileName}->{segemehl}->{-q}, '-o', $self->{fileName}->{segemehl}->{-o}, ' -F 1 2>log') ;
print STDERR join ( " ", @map),"\n" ;
system(join" ", @map) ;
# my @convertInBam = ($self->{program}->{awk}, '\'{if($6 ~/^[0-9]+M$/){print $0;} else if($1 ~/^@.*/){print $0;}}\'', $self->{fileName}->{segemehl}->{-o}, '|', $self->{program}->{samtools}, 'view -b - |', $self->{program}->{samtools},'sort - -o', $self->{fileName}->{segemehl}->{bam} ) ;
my @convertInBam = ($self->{program}->{samtools}, 'view -b', $self->{fileName}->{segemehl}->{-o}, '|', $self->{program}->{samtools},'sort - -o', $self->{fileName}->{mappingOutput}->{bam} ) ;
print STDERR join ( " ", @convertInBam),"\n" ;
system(join" ", @convertInBam) ;
my @rm = ($self->{program}->{rm}, $self->{fileName}->{segemehl}->{-o}) ;
system(join" ", @rm) ;
my @index = ($self->{program}->{samtools}, 'index ', $self->{fileName}->{mappingOutput}->{bam} ) ;
print STDERR join ( " ", @index),"\n" ;
system(join" ", @index) ;
}
sub selectPairedEndReads{
my $datestring = localtime();
print STDERR "INFO $prog $datestring Run Module: selectPairedEndReads !\n" ;
my $self = shift ;
$self->{sam} = Bio::DB::Sam->new(-bam => $self->{fileName}->{mappingOutput}->{bam} );
my $list ;
my $nbReadsStore = 0 ;
open($list, '>', $self->{option}->{unmappedFastqFile}->{prefix}.".fishReads.list") or die("DIE: Could not open file.\n");
foreach my $teid (sort keys %{$self->{targetFeat}}){
my @fam ;
if($self->{targetFeat}->{$teid}->{'te'}->has_tag('fam')){
@fam = $self->{targetFeat}->{$teid}->{'te'}->get_tag_values('fam') ;
}
else{
print STDERR "WARNING $prog: Not tag fam for feature $teid in Gff3.\n" ;
next ;
}
my $teObj = $self->{targetFeat}->{$teid}->{te} ;
my $inside = $teObj->start+$self->{option}->{maxReadsLength} ;
if($inside>$teObj->end){ $inside = $teObj->end ; }
##### for 5prime of the TEs.
my $position ;
if($teObj->start-20 < 1){
$position = $teObj->seq_id.":1-".$inside ; # select reads mapped on each TEs of interested. Position = start-20, start+200
}
else {
$position = $teObj->seq_id.":".($teObj->start-20)."-".$inside ; # select reads mapped on each TEs of interested. Position = start-20, start+200
}
$self->{sam}->fetch($position,
sub {
my $a = shift;
if($a->strand eq "1"){
my $readName = $a->display_name ;
if($readName =~/^(.*)_([0-9]+)\/2$/){
my $mate = &reverseMate($readName) ;
if(not defined $self->{targetFeat}->{$teid}->{reads}->{$mate}){
print $list $mate."\n" ;
$self->{targetFeat}->{$teid}->{reads}->{$mate} = 1 ;
}
}
}
elsif($a->strand eq "-1"){
my $readName = $a->display_name ;
if($readName =~/^(.*)_([0-9]+)\/1$/){
my $mate = &reverseMate($readName) ;
if(not defined $self->{targetFeat}->{$teid}->{reads}->{$mate}){
print $list $mate."\n" ;
$self->{targetFeat}->{$teid}->{reads}->{$mate} = 1 ;
}
}
}
}
);
#### for 3prime of the TEs.
$inside = $teObj->end-$self->{option}->{maxReadsLength} ;
if($inside < $teObj->start){ $inside = $teObj->start ; }
$position = $teObj->seq_id.":".$inside."-".($teObj->end+20) ; # select reads mapped on each TEs of interested. Position = end-200, end+50
$self->{sam}->fetch($position,
sub {
my $a = shift;
if($a->strand eq "1"){
my $readName = $a->display_name ;
if($readName =~/^(.*)_([0-9]+)\/1$/){
my $mate = &reverseMate($readName) ;
if(not defined $self->{targetFeat}->{$teid}->{reads}->{$mate}){
print $list $mate."\n" ;
$self->{targetFeat}->{$teid}->{reads}->{$mate} = 1 ;
}
}
}
elsif($a->strand eq "-1"){
my $readName = $a->display_name ;
if($readName =~/^(.*)_([0-9]+)\/2$/){
my $mate = &reverseMate($readName) ;
if(not defined $self->{targetFeat}->{$teid}->{reads}->{$mate}){
print $list $mate."\n" ;
$self->{targetFeat}->{$teid}->{reads}->{$mate} = 1 ;
}
}
}
}
);
if(defined $self->{targetFeat}->{$teid}->{reads}){
$nbReadsStore += scalar(keys %{$self->{targetFeat}->{$teid}->{reads}}) ;
}
print STDERR $teid."\t".$nbReadsStore."\r" ;
}
print STDERR "\n" ;
}
sub reverseMate{
my $readName = shift ;
my $mate ;
if($readName =~/^(.*)_([0-9]+)\/1$/){ # for h1
$mate = $1."_".$2."/2" ;
}
elsif($readName =~/^(.*)_([0-9]+)\/2$/){ # for h2
$mate = $1."_".$2."/1" ;
}
return($mate) ;
}
sub checkPairedEndPosition_v3{
my $datestring = localtime();
print STDERR "INFO $prog $datestring Run Module: checkPairedEndPosition !\n" ;
my $self = shift ;
my $list ;
open($list, '>', $self->{option}->{unmappedFastqFile}->{prefix}.'.step1.list') or die("DIE: Could not open file step1.list.\n");
foreach my $teid (sort keys %{$self->{targetFeat}}){
my @fam = $self->{targetFeat}->{$teid}->{'te'}->get_tag_values('fam') ;
my $alreadyPassed ;
READ:foreach my $readName (sort keys %{$self->{targetFeat}->{$teid}->{reads}}){
my ($read, $chopSize) = $readName =~/^(.*)_([0-9]+)\/[1-2]$/ ;
if(defined $alreadyPassed->{$read}){ next READ;}
if(defined $self->{storeReads}->{allReads}->{$readName}){ # if mate is mapped.
foreach my $num (sort keys %{$self->{storeReads}->{allReads}->{$readName}}){
if(defined $alreadyPassed->{$read}){ next READ;}
my $readChr = $self->{storeReads}->{allReads}->{$readName}->{$num}->seq_id ;
my $readStart = $self->{storeReads}->{allReads}->{$readName}->{$num}->start ;
my $readStop = $self->{storeReads}->{allReads}->{$readName}->{$num}->end ;
unless (defined $self->{bedTree}->{$fam[0]}->{$readChr}){ # read Chr different than copies Chr
print $list $read."\n" ;
if(defined $self->{storeChop}->{$read}){
if($self->{storeChop}->{$read} < $chopSize){
$self->{storeChop}->{$read} = $chopSize ;
}
}
else{
$self->{storeChop}->{$read} = $chopSize ;
}
if($chopSize == @{$self->{option}->{lengthPairedEnd}}[scalar(@{$self->{option}->{lengthPairedEnd}})-1]){
$alreadyPassed->{$read} = 1 ;
}
next ; # because $bedTree->{$readChr} is not defined in this case
};
my $overlapping = $self->{bedTree}->{$fam[0]}->{$readChr}->fetch( $readStart, $readStop );
unless(defined $overlapping and @$overlapping){ # read Chr is not overlapping copies
print $list $read."\n" ;
if(defined $self->{storeChop}->{$read}){
if($self->{storeChop}->{$read} < $chopSize){
$self->{storeChop}->{$read} = $chopSize ;
}
}
else{
$self->{storeChop}->{$read} = $chopSize ;
}
if($chopSize == @{$self->{option}->{lengthPairedEnd}}[scalar(@{$self->{option}->{lengthPairedEnd}})-1]){
$alreadyPassed->{$read} = 1 ;
}
}
}
}
else{ # the other mate isn't mapped => keep it.
print $list $read."\n" ;
$alreadyPassed->{$read} = 1 ;
}
}
delete($self->{targetFeat}->{$teid}->{reads}) ;
}
$self->{storeReads} = () ;
}
sub checkPairedEndPosition_v2{
my $datestring = localtime();
print STDERR "INFO $prog $datestring Run Module: checkPairedEndPosition !\n" ;
my $self = shift ;
my $list ;
open($list, '>', $self->{option}->{unmappedFastqFile}->{prefix}.'.step1.list') or die("DIE: Could not open file step1.list.\n");
foreach my $teid (sort keys %{$self->{targetFeat}}){
my @fam = $self->{targetFeat}->{$teid}->{'te'}->get_tag_values('fam') ;
my $alreadyPassed ;
READ:foreach my $readName (sort keys %{$self->{targetFeat}->{$teid}->{reads}}){
my ($read) = $readName =~/^(.*)_[0-9]+\/[1-2]$/ ;
if(defined $alreadyPassed->{$read}){ next READ;}
if(defined $self->{storeReads}->{allReads}->{$readName}){ # if mate is mapped.
foreach my $num (sort keys %{$self->{storeReads}->{allReads}->{$readName}}){
if(defined $alreadyPassed->{$read}){ next READ;}
my $readChr = $self->{storeReads}->{allReads}->{$readName}->{$num}->seq_id ;
my $readStart = $self->{storeReads}->{allReads}->{$readName}->{$num}->start ;
my $readStop = $self->{storeReads}->{allReads}->{$readName}->{$num}->end ;
unless (defined $self->{bedTree}->{$fam[0]}->{$readChr}){ # read Chr different than copies Chr
print $list $read."\n" ;
$alreadyPassed->{$read} = 1 ;
next ; # because $bedTree->{$readChr} is not defined in this case
};
my $overlapping = $self->{bedTree}->{$fam[0]}->{$readChr}->fetch( $readStart, $readStop );
unless(defined $overlapping and @$overlapping){ # read Chr is not overlapping copies
print $list $read."\n" ;
$alreadyPassed->{$read} = 1 ;
}
}
}
else{ # the other mate isn't mapped => keep it.
print $list $read."\n" ;
$alreadyPassed->{$read} = 1 ;
}
}
delete($self->{targetFeat}->{$teid}->{reads}) ;
}
$self->{storeReads} = () ;
}
sub pairedEndGrab {
my $datestring = localtime();
print STDERR "INFO $prog $datestring Run Module: pairedEndGrab !\n" ;
my $self = shift;
my $sam ;
my @features;
my $fileName = $self->{option}->{unmappedFastqFile}->{prefix}.".matePairedEnd.bam" ;
my @bamutils = ($self->{program}->{bamutils}, 'filter ', $self->{fileName}->{mappingOutput}->{bam} , $fileName, '-whitelist ', $self->{option}->{unmappedFastqFile}->{prefix}.'.fishReads.list') ;
print STDERR join ( " ", @bamutils),"\n" ;
system(join" ", @bamutils) ;
my @index = ($self->{program}->{samtools},'index', $fileName) ;
print STDERR join ( " ", @index),"\n" ;
system(join" ", @index) ;
$sam = Bio::DB::Sam->new(-bam => $fileName);
@features = $sam->features("match") ; # output all matching reads.
for my $a (@features){
if(not defined $self->{storeReads}->{allReads}->{$a->display_name}){
$self->{storeReads}->{allReads}->{$a->display_name}->{1} = $a ;
}
elsif(defined $self->{storeReads}->{allReads}->{$a->display_name}){
my $num = scalar (keys %{$self->{storeReads}->{allReads}->{$a->display_name}})+1 ;
$self->{storeReads}->{allReads}->{$a->display_name}->{$num} = $a ;
}
}
}
sub selectSplitReads_v2{
my $datestring = localtime();
print STDERR "INFO $prog $datestring Run Module: selectSplitReads !\n" ;
my $self = shift;
$self->{sam} = Bio::DB::Sam->new(-bam => $self->{fileName}->{mappingOutput}->{bam} ,
-fasta => $self->{fileName}->{refFasta}->{file});
# select the bam file header to later print bam.
if(not defined $self->{printBam}->{header}){
my $bam = Bio::DB::Bam->open($self->{fileName}->{mappingOutput}->{bam}) ;
$self->{printBam}->{header} = $bam->header() ;
}
my $list ;
my $passed ;
open($list, '>', $self->{option}->{unmappedFastqFile}->{prefix}.".fishReads.list") or die("DIE: Could not open file.\n");
foreach my $teid (sort keys %{$self->{targetFeat}}){
print STDERR $teid."\r" ;
my $teObj = $self->{targetFeat}->{$teid}->{te} ;
my @fam = $self->{targetFeat}->{$teid}->{'te'}->get_tag_values('fam') ;
##### for 5prime of the TEs.
my $position = $teObj->seq_id.":".($teObj->start)."-".($teObj->start+$self->{option}->{maxReadsLength}) ;
$self->{sam}->fetch($position,
sub {
my $a = shift;
my ($readId, $halfRead, $size, $splitPosition, $break, $fullReadLength) = $self->spliceReadsId($a->display_name) ;
if($halfRead == 1){ # for h1
if($a->strand == "-1"){ # if h1 and strand = + => useless
# selected mNeoReadId
for(my $i = $break ; $i<$break+10 ; $i++){
my $mNeoReadId = $readId."_".$i."_".$fullReadLength."/2" ;
if(not defined $passed->{$mNeoReadId}){
print $list $mNeoReadId."\n" ;
}
$passed->{$mNeoReadId} = 1 ;
}
# load $self->{nbTimeMapped} for multi-mapped reads
if(defined $self->{nbTimeMapped}->{mTE}->{$a->display_name}){
$self->{nbTimeMapped}->{mTE}->{$a->display_name} += 1 ;
}
else{
$self->{nbTimeMapped}->{mTE}->{$a->display_name} = 1 ;
}
$self->{reads}->{mTE}->{$self->{nbTimeMapped}->{mTE}->{$a->display_name}."--".$a->display_name}->{subPart} = 1 ;
$self->{reads}->{mTE}->{$self->{nbTimeMapped}->{mTE}->{$a->display_name}."--".$a->display_name}->{featureID} = $teid ;
$self->{reads}->{mTE}->{$self->{nbTimeMapped}->{mTE}->{$a->display_name}."--".$a->display_name}->{fam} = $fam[0] ;
$self->{reads}->{mTE}->{$self->{nbTimeMapped}->{mTE}->{$a->display_name}."--".$a->display_name}->{obj} = $a ;
# exact insertion
if($teObj->start == $a->start){ # read mapped exactly at the start
if($teObj->strand == 1){
$self->{reads}->{mTE}->{$self->{nbTimeMapped}->{mTE}->{$a->display_name}."--".$a->display_name}->{mappedInStart} = 1 ;
}
elsif($teObj->strand == 1){
$self->{reads}->{mTE}->{$self->{nbTimeMapped}->{mTE}->{$a->display_name}."--".$a->display_name}->{mappedInStart} = 1 ;
}
$self->{mappedOnStartEnd}->{$readId}->{$splitPosition} = 1 ;
}
# determine mtExtremity
if($teObj->strand == 1){
$self->{reads}->{mTE}->{$self->{nbTimeMapped}->{mTE}->{$a->display_name}."--".$a->display_name}->{mtExtremity} = 5 ;
}
elsif($teObj->strand == -1){
$self->{reads}->{mTE}->{$self->{nbTimeMapped}->{mTE}->{$a->display_name}."--".$a->display_name}->{mtExtremity} = 3 ;
}
}
}
elsif($halfRead == 2){ # for h2
if($a->strand == "+1"){ # if h2 and strand = - => useless
# selected mNeoReadId
for(my $i = $break ; $i>$break-10 ; $i--){
my $mNeoReadId = $readId."_".$i."_".$fullReadLength."/1" ;
if(not defined $passed->{$mNeoReadId}){
print $list $mNeoReadId."\n" ;
}
$passed->{$mNeoReadId} = 1 ;
}
# load $self->{nbTimeMapped}
if(defined $self->{nbTimeMapped}->{mTE}->{$a->display_name}){
$self->{nbTimeMapped}->{mTE}->{$a->display_name} += 1 ;
}
else{
$self->{nbTimeMapped}->{mTE}->{$a->display_name} = 1 ;
}
$self->{reads}->{mTE}->{$self->{nbTimeMapped}->{mTE}->{$a->display_name}."--".$a->display_name}->{subPart} = 2 ;
$self->{reads}->{mTE}->{$self->{nbTimeMapped}->{mTE}->{$a->display_name}."--".$a->display_name}->{featureID} = $teid ;
$self->{reads}->{mTE}->{$self->{nbTimeMapped}->{mTE}->{$a->display_name}."--".$a->display_name}->{fam} = $fam[0] ;
$self->{reads}->{mTE}->{$self->{nbTimeMapped}->{mTE}->{$a->display_name}."--".$a->display_name}->{obj} = $a ;
# exact insertion
if($teObj->start == $a->start){ # read mapped exactly at the start
if($teObj->strand == 1){
$self->{reads}->{mTE}->{$self->{nbTimeMapped}->{mTE}->{$a->display_name}."--".$a->display_name}->{mappedInStart} = 1 ;
}
elsif($teObj->strand == 1){
$self->{reads}->{mTE}->{$self->{nbTimeMapped}->{mTE}->{$a->display_name}."--".$a->display_name}->{mappedInEnd} = 1 ;
}
$self->{mappedOnStartEnd}->{$readId}->{$splitPosition} = 1 ;
}
if($teObj->strand == 1){
$self->{reads}->{mTE}->{$self->{nbTimeMapped}->{mTE}->{$a->display_name}."--".$a->display_name}->{mtExtremity} = 5 ;
}
elsif($teObj->strand == -1){
$self->{reads}->{mTE}->{$self->{nbTimeMapped}->{mTE}->{$a->display_name}."--".$a->display_name}->{mtExtremity} = 3 ;
}
}
}
});
#### for 3prime of the TEs.
$position = $teObj->seq_id.":".($teObj->end-$self->{option}->{maxReadsLength})."-".$teObj->end ;
$self->{sam}->fetch($position,
sub {
my $a = shift;
my ($readId, $halfRead, $size, $splitPosition, $break, $fullReadLength) = $self->spliceReadsId($a->display_name) ;
if($halfRead == 1){ # for h1
if($a->strand == "1"){ # if h1 and strand = - => useless
# selected mNeoReadId
for(my $i = $break ; $i<$break+10 ; $i++){
my $mNeoReadId = $readId."_".$i."_".$fullReadLength."/2" ;
if(not defined $passed->{$mNeoReadId}){
print $list $mNeoReadId."\n" ;
}
$passed->{$mNeoReadId} = 1 ;
}
# load $self->{nbTimeMapped}
if(defined $self->{nbTimeMapped}->{mTE}->{$a->display_name}){
$self->{nbTimeMapped}->{mTE}->{$a->display_name} += 1 ;
}
else{
$self->{nbTimeMapped}->{mTE}->{$a->display_name} = 1 ;
}
$self->{reads}->{mTE}->{$self->{nbTimeMapped}->{mTE}->{$a->display_name}."--".$a->display_name}->{subPart} = 1 ;
$self->{reads}->{mTE}->{$self->{nbTimeMapped}->{mTE}->{$a->display_name}."--".$a->display_name}->{featureID} = $teid ;
$self->{reads}->{mTE}->{$self->{nbTimeMapped}->{mTE}->{$a->display_name}."--".$a->display_name}->{fam} = $fam[0] ;
$self->{reads}->{mTE}->{$self->{nbTimeMapped}->{mTE}->{$a->display_name}."--".$a->display_name}->{obj} = $a ;
# exact insertion
if($teObj->end == $a->end){ # read mapped exactly at the start
if($teObj->strand == 1){
$self->{reads}->{mTE}->{$self->{nbTimeMapped}->{mTE}->{$a->display_name}."--".$a->display_name}->{mappedInEnd} = 1 ;
}
elsif($teObj->strand == 1){
$self->{reads}->{mTE}->{$self->{nbTimeMapped}->{mTE}->{$a->display_name}."--".$a->display_name}->{mappedInStart} = 1 ;
}
$self->{mappedOnStartEnd}->{$readId}->{$splitPosition} = 1 ;
}
if($teObj->strand == 1){
$self->{reads}->{mTE}->{$self->{nbTimeMapped}->{mTE}->{$a->display_name}."--".$a->display_name}->{mtExtremity} = 3 ;
}
elsif($teObj->strand == -1){
$self->{reads}->{mTE}->{$self->{nbTimeMapped}->{mTE}->{$a->display_name}."--".$a->display_name}->{mtExtremity} = 5 ;
}
}
}
elsif($halfRead == 2){ # for h2
if($a->strand == "-1"){ # if h2 and strand = + => useless
# selected mNeoReadId
for(my $i = $break ; $i>$break-10 ; $i--){
my $mNeoReadId = $readId."_".$i."_".$fullReadLength."/1" ;
if(not defined $passed->{$mNeoReadId}){
print $list $mNeoReadId."\n" ;
}
$passed->{$mNeoReadId} = 1 ;
}