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#!/usr/bin/env perl
###################################################################################################################
#Marten Boetzer BaseClear B.v. 14-07-2011 #
#SSPACE perl subscript samToTab_multi.pl #
#This script; #
# -Estimates median insert size by mapping paired-reads on contigs #
# It goes through each contig and maps both reads, if a pair is mapped, #
# the orientation and insert size is estimated. #
# If sufficient pairs (given by the user) are found, the median insert size is #
# estimated, as well as a file with the distribution is generated which can be #
# used to visualize the insert size distribution. #
# #
# To run this script; #
# perl estimate_insert_size.pl <contigfile> <readfile1> <readfile2> <number_of_pairs> <orientation_of_pairs> #
#
# Output is the median insert size and a file with distribution of the insert size. Also, number of pairs for #
# each found orientation (FR, RF, FF and RR) are given. #
###################################################################################################################
use File::Path;
use strict;
my $contigfile = $ARGV[0];
my $fileA = $ARGV[1];
my $fileB = $ARGV[2];
my $numpairs = $ARGV[3];
my $orientation = $ARGV[4];
die "ERROR: Can't find contig file: $contigfile -- fatal\n" if(! -e $contigfile);
die "ERROR: Can't find read file: $fileA -- fatal\n" if(! -e $fileA);
die "ERROR: Can't find read file: $fileB -- fatal\n" if(! -e $fileB);
if($numpairs eq ''){
print "WARNING: No number of pairs are given, using 10000 pairs instead\n";
$numpairs = 10000;
}
if($orientation eq ''){
print "WARNING: No orientation of the pairs is given, using orientation FR instead\n";
$orientation = "FR";
}
die "ERROR: You've inserted $numpairs, which does not seem to be an valid number. Exiting.\n" if(!($numpairs>0) || !($numpairs =~ /^\d+$/));
die "ERROR: Orientation must have length of 2 characters and should contain one of the following; FR, FF, FR or RF. You've inserted orientation of $orientation ...Exiting.\n" if(!(length($orientation) == 2) || !($orientation =~ /[FR][FR]/));
print "\n";
my $paircount = 0;
my ($direction, $insertsize);
mkpath('bowtieoutput');
open (CONT, $contigfile) || die "Can't open contig file $contigfile\n";
my ($seq,$name, $maxctg, $maxseq, $maxname)=("","",0,"","");
my $contignum = 0;
CONTIG:
while (<CONT>) {
chomp;
$seq.=$_ if(eof(CONT));
if (/\>(\S+)/ || eof(CONT)){
if($seq ne ""){
$contignum++;
if(length($seq) > $maxctg){
$maxctg = length($seq);
$maxseq = $seq;
$maxname = $name;
}
if(eof(CONT)){
$seq = $maxseq;
$name = $maxname;
}
if(eof(CONT)){
print "now at contig $name = size".length($seq)."\n";
open (BOWCONT, ">bowtieoutput/bowtie_input.fa");
print BOWCONT ">$name\n$seq\n";
close BOWCONT;
($paircount) = &mapWithBowtie($contignum,"bowtieoutput/bowtie_input.fa", $fileA, $fileB);
last CONTIG if($paircount>=$numpairs);
}
$name = "";
$seq = "";
}
$name = $1;
}
else {
$seq .= $_;
}
}
foreach my $d (keys %$direction){
print "direction $d is found $direction->{$d} times\n";
}
my ($median_ins,$record) = (0,0);
my $median_bin = int($paircount/2);
open (CSV, ">distribution.txt") || die "Can't open distribution.txt for writing -- fatal";
foreach my $is (sort {$a<=>$b} keys %$insertsize){
for(my $i=0;$i<$insertsize->{$is};$i++){
$record++;
$median_ins = $is if($record >= $median_bin && $median_ins == 0);
}
print CSV "$is\t$insertsize->{$is}\n";
}
print "\nmedian = $median_ins\n\nSee the distribution in file 'distribution.txt'\n";
sub mapWithBowtie{
my ($fname,$contig, $fileA, $fileB) = @_;
my $bowtieout = "contig$fname.bowtieIndex";
system("bowtie-build $contig bowtieoutput/$bowtieout --quiet --noref") == 0 || die "\nBowtie-build error; $?"; # returns exit status values
my $fastq = 0;
open(TEST, "< $fileA");
$name = <TEST>;
close TEST;
$fastq = 1 if ($name =~ /^[@]/);
open(FILEA, "< $fileA");
open(FILEB, "< $fileB");
my $count=0;
open (BOWIN, ">bowtieoutput/bowtiein.$fname.fa") || die "Can't write to single file bowtieoutput/bowtiein.$fname.fa-- fatal\n";
while(<FILEA>) {
<FILEB>;
$count++;
my $seq1 = <FILEA>;
chomp $seq1;
my $seq2 = <FILEB>;
chomp $seq2;
#FASTQ FORMAT
<FILEA>,<FILEA>,<FILEB>,<FILEB> if ($fastq);
print BOWIN ">read$count\n$seq1>read$count\n$seq2";
if($count > $numpairs){
close BOWIN;
open(IN, "bowtie -p 1 -v 0 -m 1 bowtieoutput/$bowtieout --suppress 6,7 -f bowtieoutput/bowtiein.$fname.fa --quiet|") || die "Can't open bowtie output -- fatal\n";
my ($prevread, $prevline);
while(my $line = <IN>){
my @t1 = split(/\t/,$line);
if($prevread eq $t1[0]){
$paircount++;
my @t2 = split(/\t/,$prevline);
my ($start1, $start2, $end1,$end2);
if($t1[1] eq "+"){
$end1 = $t1[3] + length($t1[4]);
$start1 = $t1[3];
}
else{
$start1 = $t1[3] + length($t1[4]);
$end1 = $t1[3];
}
if($t2[1] eq "+"){
$end2 = $t2[3] + length($t2[4]);
$start2 = $t2[3];
}
else{
$start2 = $t2[3] + length($t2[4]);
$end2 = $t2[3];
}
my ($dir1, $dir2);
$dir1 = "F" if($start1 < $end1);
$dir1 = "R" if($start1 > $end1);
$dir2 = "F" if($start2 < $end2);
$dir2 = "R" if($start2 > $end2);
$direction->{"$dir1$dir2"}++ if($start1 < $start2);
$direction->{"$dir2$dir1"}++ if($start2 < $start1);
my $diff = abs($start2-$start1);
if($orientation eq "$dir1$dir2" || $orientation eq "$dir2$dir1"){
$insertsize->{$diff}++;
}
return $paircount if($paircount >= $numpairs);
}
$prevread = $t1[0];
$prevline = $line;
}
close BOWIN;
open (BOWIN, "bowtieoutput/bowtiein.$fname.fa") || die "Can't write to single file bowtieoutput/bowtiein.$name.fa-- fatal\n";
}
}
print "count = $paircount\n";
return $paircount;
}
###PRINTS A COUNTER ON THE SCREEN AND OVERWRITES PREVIOUS LINE
sub CounterPrint{
my $countingMessager = shift;
print "\r$countingMessager";
$|++;
}
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