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#!/usr/bin/perl
#
# Map a sam file that was created by aligning against an exon-exon junction file back to genome level.
# Filters out non-intron-containing alignments.
#
# Katharina J. Hoff, May 3rd 2012
#use strict;
#use Getopt::Long;
my $usage = "\n$0 -- Map a sam file that was created by aligning against an exon-exon junction file back to genome level\n\n";
$usage .= "Usage: $0 exex.sam map.psl [flank] > out.sam\n\n";
$usage .= "exex.sam was created e.g. with GSNAP in the following way:\n";
$usage .= "gmap_build -d exex_db exex.fa\n";
$usage .= "gsnap --format=sam --nofails -d exex_db rnaseq.fastq > exex.sam\n\n";
$usage .= "map.psl was created in the following way:\n";
$usage .= "intron2exex.pl --introns=introns.lst --seq=genome.masked.fa --exex=exex.fa --map=map.psl\n\n";
$usage .= "Please note that mapSam.pl assumes that the flanking region of introns is 75+75 bp! Specify other value if needed.\n";
if(! defined $ARGV[1]){
print $usage;
exit;
}
my $exexFile = $ARGV[0];
my $mapFile = $ARGV[1];
my $flank = 75;
if(defined $ARGV[2]){
$flank = $ARGV[2];
}
my %mapHash = ();
my @t;
# read map file into hash, lookup by exexID
open(MAP, "<", $mapFile) or die("Could not open mapFile $mapFile!\n");
while(<MAP>){
@t = split(/\t/);
if(not(exists($mapHash{$t[9]}))){
$mapHash{$t[9]} = $_;
}else{
print STDERR "Hash element $t[9] in mapHash already exists!\n";
}
}
close(MAP) or die("Could not close mapFile $mapFile!\n");
my @tSam;
my $readLen;
my $exexName;
my $mapLine;
my @tMap;
my $junctionStart;
my $beforeJunction;
my $globalStart;
my $cigarLen;
my $cigPos;
my $safeForAfterJunctionNum;
my $safeForAfterJunctionDesc;
my $seenJunction;
my $intronLen;
my $samLen;
my $letter;
my $number;
my $newCigar;
my $startCoord;
my $cigarNumber;
my $badCigar = 0;
# process each line of sam format
open(SAM, "<", $exexFile) or die("Could not open exexFile $exexFile!\n");
while(<SAM>){
if(not(m/^@/)){
@tSam = split(/\t/);
$readLen = length($tSam[9]);
$exexName = $tSam[2];
$mapLine = $mapHash{$exexName};
@tMap = split(/\t/, $mapLine);
$junctionStart = $tMap[15]+$tMap[0]/2+1; # first base after
if(($tMap[0]/2)==$flank){ # results from fragment border introns!
$globalStart = $tSam[3]+$tMap[15];
$intronLen = $tMap[7];
# process the cigar string
my $softclipped = 0;
if($tSam[5]=~m/^(\d+)S/){
$softclipped = $1;
}
$beforeJunction = $junctionStart - $globalStart + $softclipped;
if($beforeJunction > 0){
$startCoord = $tSam[3]+$tMap[15];
}else{
$startCoord = ($tSam[3]+$tMap[15]+$tMap[7]);
}
$cigarLen = length($tSam[5]);
$safeForAfterJunction = 0;
$cigPos = 1;
$seenJunction = 0;
$newCigar = "";
while($cigarLen > 0){
$tSam[5] =~ m/^(\d+)(\w)/;
$letter = $2;
$number = $1;
if($tSam[3]>($tMap[0]/2)){
$seenJunction = 1;
}
if($number <= $beforeJunction and $seenJunction == 0){
$cigarNumber = $number;
if(($cigarNumber==0 and $letter eq 'M') or $cigarNumber=~m/\D/){
$badCigar = 1;
}
$newCigar = $newCigar.$cigarNumber.$letter;
$beforeJunction = $beforeJunction - $number;
}elsif($seenJunction == 0 and $beforeJunction >= 1 ){
$cigarNumber = ($number - ($number - $beforeJunction));
if(($cigarNumber==0 and $letter eq 'M') or $cigarNumber=~m/\D/){
$badCigar = 1;
}
$newCigar = $newCigar.$cigarNumber;
$newCigar = $newCigar.$letter;
$newCigar = $newCigar.$intronLen."N";
$cigarNumber = ($number-$beforeJunction);
if(($cigarNumber==0 and $letter eq 'M') or $cigarNumber=~m/\D/){
$badCigar = 1;
}
$newCigar = $newCigar.$cigarNumber;
$newCigar = $newCigar.$letter;
$seenJunction = 1;
}else{
$cigarNumber = $number;
if(($cigarNumber==0 and $letter eq 'M') or $cigarNumber=~m/\D/){
$badCigar = 1;
}
$newCigar = $newCigar.$cigarNumber.$letter;
}
$cigPos = $cigPos + $number;
$tSam[5]=~ s/$number$letter//;
$cigarLen = length($tSam[5]);
}
if($newCigar =~ m/N/ and $badCigar==0 and not($newCigar=~m/\d+H\d+N\d+H/)){
print $tSam[0]."\t".$tSam[1]."\t".$tMap[13]."\t".$startCoord."\t".$tSam[4]."\t$newCigar";
$samLen = @tSam;
for ($count = 6; $count < $samLen; $count ++){
print "\t$tSam[$count]";
}
}elsif($badCigar==1 or $newCigar=~m/\d+H\d+N\d+H/){
print STDERR "Warning - Bad CIGAR (this alignment is not included in the output at stdout):\n".$tSam[0]."\t".$tSam[1]."\t".$tMap[13]."\t".$startCoord."\t".$tSam[4]."\t$newCigar\n";
$samLen = @tSam;
for ($count = 6; $count < $samLen; $count ++){
print STDERR "\t$tSam[$count]";
}
print STDERR "Sam:\n";
print STDERR $_;
print STDERR "Map:\n";
foreach(@tMap){
print STDERR "\t".$_;
}
}
$badCigar = 0;
}
}
}
close(SAM) or die("Could not close exexFile $exexFile!\n");
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