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#!/usr/bin/env perl
# NOTE FOR WEB GUIDE http://trinityrnaseq.sourceforge.net/genome_guided_trinity.html
# % samtools view gsnap_out/gsnap.coordSorted.bam > gsnap.coordSorted.sam
# change to run samtools view with -F4 or -F12 if you are plannign to use --no_single
use strict;
use warnings;
use lib ("/usr/lib/trinityrnaseq/PerlLib");
use SAM_reader;
use SAM_entry;
use COMMON;
use Getopt::Long qw(:config no_ignore_case bundling);
use Carp;
use Data::Dumper;
use Cwd;
$ENV{LC_ALL} = 'C'; # critical for proper sorting using [system "sort -k1,1 ..."] within the perl script
my $usage = <<_EOUSAGE_;
#######################################################################
#
# Required:
#
# --sam <string> SAM-formatted file
#
# Optional:
#
# --no_single no single reads
#
# --min_insert_size <int> minimum distance between proper pair's starting positions. (default: 100)
# --max_insert_size <int> maximum distance between proper pair's starting positions. (default: 500)
#
# --sort_buffer <string> default '10G', amount of RAM to allocate to sorting
#
# -d debug mode
#
#######################################################################
_EOUSAGE_
;
my $sam_file;
my $full_flag = 0;
my $MAX_INSERT_SIZE = 500;
my $MIN_INSERT_SIZE = 100;
my $DEBUG = 0;
my $help_flag = 0;
my $NO_SINGLE;
my $CPU = 1;
my $sort_buffer = '10G';
&GetOptions(
'h' => \$help_flag,
'sam=s' => \$sam_file,
'CPU=i' => \$CPU,
'sort_buffer=s' => \$sort_buffer,
'no_single' => \$NO_SINGLE,
'max_insert_size=i' => \$MAX_INSERT_SIZE,
'min_insert_size=i' => \$MIN_INSERT_SIZE,
'd' => \$DEBUG,
);
if ($help_flag) {
die $usage;
}
unless ($sam_file) {
die $usage;
}
my $sort_exec = &COMMON::get_sort_exec($CPU);
main: {
my @frags;
my $read_coords_file = "$sam_file.read_coords";
my $read_coords_checkpoint = "$read_coords_file.ok";
&extract_read_coords($read_coords_file) unless (-s $read_coords_checkpoint);
&process_cmd("touch $read_coords_checkpoint");
my $pair_coords_file = "$sam_file.frag_coords";
&extract_frag_coords($read_coords_file, $pair_coords_file);
exit(0);
}
####
sub extract_read_coords {
my ($read_coords_file) = @_;
# AP: I think this is very expensive (more than 20' for 12G SAM)
# option 1) the same information could be derived by parsing the BAM file
# and cut -f 1,2,3,7,8,4,9,10 -> there will be two identical (1) fields, if paired the one with (9)>0 is the /1 one||the one on the plus strand (from (2)) could be assigned as the /1 one
# generally, i don't see the need to have to create a SAM file in the first place...
# another benefit of samtools view bam <region> is that we could extract each scaffold separately (would help with sort...)
# option 2) use samtools depth instead of wig file???
## Every read processed.
print STDERR "-extracting read coordinates from $sam_file into $read_coords_file\n\n";
my $sam_reader = new SAM_reader($sam_file);
open (my $ofh, ">$read_coords_file") or die "Error, cannot write to file $read_coords_file";
while ($sam_reader->has_next()) {
my $sam_entry = $sam_reader->get_next();
# unless ($sam_entry->get_mate_scaffold_name() eq "=" || $sam_entry->get_mate_scaffold_name() eq $sam_entry->get_scaffold_name()) { next; }
# commenting out above - just describe the reads, let the other routine handle the fragment definition.
eval {
my $scaffold = $sam_entry->get_scaffold_name();
my $core_read_name = $sam_entry->get_core_read_name();
my $read_name = $sam_entry->get_read_name();
my $full_read_name = $sam_entry->reconstruct_full_read_name();
#print "read_name: $read_name, full_read_name: $full_read_name\n";
my $pair_side = ".";
if ($full_read_name =~ m|/([12])$|) {
$pair_side = $1;
}
elsif ($read_name =~ m|/([12])$|) {
$pair_side = $1;
}
my $mate_scaff_pos = $sam_entry->get_mate_scaffold_position();
my ($read_start, $read_end) = $sam_entry->get_genome_span();
print $ofh join("\t", $scaffold, $core_read_name, $pair_side, $read_start, $read_end) . "\n" if $scaffold ne '*' && $read_start && $read_end;
};
if ($@) {
print STDERR "******\nError parsing SAM entry: " . Dumper($sam_entry) . " \n$@\n******\n\n\n";
}
}
close $ofh;
return;
}
####
sub extract_frag_coords {
my ($read_coords_file, $pair_frag_coords_file) = @_;
unless (-s "$read_coords_file.sort_by_readname" ){
## sort by scaffold, then by read name
my $cmd = "$sort_exec -S$sort_buffer -T . -k1,1 -k2,2 -k4,4n $read_coords_file > $read_coords_file.sort_by_readname";
&process_cmd($cmd);
rename("$read_coords_file.sort_by_readname", $read_coords_file);
# so that sort is not re-done...
#symlink(&create_full_path($read_coords_file),&create_full_path("$read_coords_file.sort_by_readname"));
&process_cmd("cp " . &create_full_path($read_coords_file) . " " . &create_full_path("$read_coords_file.sort_by_readname")); # some systems dont like symlinks
}
## define fragment pair coordinate span
open (my $fh, "$read_coords_file") or die $!;
open (my $ofh, ">$pair_frag_coords_file") or die $!;
my $prev_reported_pair = "";
my $prev_reported_single = "";
my $first = <$fh>;
chomp $first;
while (my $second = <$fh>) {
next if ($first =~/^\*/ && $second =~/^\*/); # both unmapped
chomp $second;
my ($scaffA, $readA, $readA_pair_side, $lendA, $rendA) = split(/\t/, $first);
my ($scaffB, $readB, $readB_pair_side, $lendB, $rendB) = split(/\t/, $second);
my $got_pair_flag = 0;
if ($readA eq $readB
&& $scaffA eq $scaffB
&& $readA_pair_side ne $readB_pair_side
&& $readA_pair_side =~ /\d/ && $readB_pair_side =~ /\d/) {
my @coords = sort {$a<=>$b} ($lendA, $rendA, $lendB, $rendB);
my $min = shift @coords;
my $max = pop @coords;
my $insert_size = $max - $min + 1;
if ($insert_size >= $MIN_INSERT_SIZE && $insert_size <= $MAX_INSERT_SIZE && $readA ne $prev_reported_pair) {
# treat as proper pair
print $ofh join("\t", $scaffA, $readA, $min, $max) . "\n";
$prev_reported_pair = $readA; # only one proper pair to be reported. - not random though, first one encountered.
}
else {
# treat as unpaired reads
unless ($NO_SINGLE) {
if ($prev_reported_single ne $readA) {
print $ofh join("\t", $scaffA, $readA . "/$readA_pair_side", $lendA, $rendA) . "\n";
print $ofh join("\t", $scaffB, $readB . "/$readB_pair_side", $lendB, $rendB) . "\n";
}
$prev_reported_single = $readA;
}
}
$first = <$fh>; # prime first
chomp $first if $first;
}
else {
# not paired
unless ($NO_SINGLE) {
if ($prev_reported_single ne $readA) {
print $ofh join("\t", $scaffA, $readA . "/$readA_pair_side", $lendA, $rendA) . "\n";
}
$prev_reported_single = $readA; # only letting one slip through.
}
$first = $second;
next;
}
}
close $ofh;
close $fh;
my $cmd = "$sort_exec -S$sort_buffer -T . -k1,1 -k3,3n $pair_frag_coords_file > $pair_frag_coords_file.coord_sorted";
&process_cmd($cmd) unless -s "$pair_frag_coords_file.coord_sorted";
rename("$pair_frag_coords_file.coord_sorted", $pair_frag_coords_file);
return;
}
####
sub process_cmd {
my ($cmd) = @_;
print STDERR "CMD: $cmd\n";
my $ret = system($cmd);
if ($ret) {
die "Error, cmd: $cmd died with ret $ret";
}
return;
}
####
sub create_full_path {
my ($path) = @_;
unless ($path =~ /^\//) {
$path = cwd() . "/$path";
}
return($path);
}
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