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#!/usr/bin/env perl
use strict;
use warnings;
use lib ("/usr/lib/trinityrnaseq/PerlLib");
use SAM_entry;
my $usage = "\n\nusage: $0 nameSorted.sam iworm_fasta_file\n\n";
my $name_sorted_sam_file = $ARGV[0] or die $usage;
my $iworm_fasta = $ARGV[1] or die $usage;
my $SAM_OFH;
main: {
open ($SAM_OFH, ">scaffolding_entries.sam") or die $!;
my %iworm_acc_to_fasta_index;
{
my $counter = 0;
open (my $fh, $iworm_fasta) or die "Error, cannot open file $iworm_fasta";
while (<$fh>) {
if (/^>(\S+)/) {
my $acc = $1;
$iworm_acc_to_fasta_index{$acc} = $counter; # starts at zero
$counter++;
}
}
close $fh;
}
my %paired_iworm_contigs;
my $prev_core_acc = "";
my %end_to_iworm;
my $num_warnings = 0;
my $num_unrecognized_iworm_contig_names = 0;
if ($name_sorted_sam_file =~ /\.bam$/) {
$name_sorted_sam_file = "samtools view $name_sorted_sam_file | ";
}
open (my $fh, $name_sorted_sam_file) or die "Error, cannot open file $name_sorted_sam_file";
while (<$fh>) {
my $line = $_;
chomp;
my @x = split(/\t/);
my $read_acc = $x[0];
my $iworm_acc = $x[2];
unless (defined $iworm_acc) { next; }
if ($iworm_acc eq '*') {
# unmapped read
next;
}
unless ($iworm_acc =~ /^a\d+;\d+/) {
$num_unrecognized_iworm_contig_names++;
if ($num_unrecognized_iworm_contig_names <= 10) {
print STDERR "warning, inchworm contig ($iworm_acc) isn't recognized. Ignoring entry: [[$line]]\n";
}
if ($num_unrecognized_iworm_contig_names == 11) {
print STDERR "warning, too many unrecognized inchworm contig names. Will report summary of counts later.\n";
}
next;
}
my $core_acc;
my $frag_end;
if ($read_acc =~ /^(\S+)\/([12])$/) {
$core_acc = $1;
$frag_end = $2;
}
# capture long read mappings
elsif ($read_acc =~ /^(LR\$\|\S+)/) {
$core_acc = $1;
$frag_end = "LR";
}
else {
# must have mixed in a single read with the pairs...
if ($num_warnings <= 10) {
print STDERR "warning, ignoring read: $read_acc since cannot decipher if /1 or /2 of a pair.\n";
}
elsif ($num_warnings == 11) {
print STDERR "number of read warnings exceeded 10. Turning off warning messages from here out.\n";
}
$num_warnings++;
next;
}
if ($core_acc ne $prev_core_acc) {
&examine_frags(\%end_to_iworm, \%paired_iworm_contigs);
%end_to_iworm = ();
}
$end_to_iworm{$frag_end}->{$iworm_acc} = $line;
$prev_core_acc = $core_acc;
}
close $fh;
## get last one
&examine_frags(\%end_to_iworm, \%paired_iworm_contigs);
if ($num_warnings) {
print STDERR "WARNING: note there were $num_warnings reads that could not be deciphered as being /1 or /2 of a PE fragment. Hopefully, these were SE reads that should have been ignored. Otherwise, please research this further.\n\n";
}
if ($num_unrecognized_iworm_contig_names) {
print STDERR "WARNING: note, there were $num_unrecognized_iworm_contig_names inchworm contig names in the SAM file that were ignored due to the inchworm contig accession not being recognized.\n";
}
foreach my $pairing (reverse sort {$paired_iworm_contigs{$a}<=>$paired_iworm_contigs{$b}} keys %paired_iworm_contigs) {
my ($iworm_acc_A, $iworm_acc_B) = split(/\t/, $pairing);
my $index_A = $iworm_acc_to_fasta_index{$iworm_acc_A};
unless (defined $index_A) {
print STDERR "WARNING, no index for iworm acc: $iworm_acc_A\n";
next;
}
my $index_B = $iworm_acc_to_fasta_index{$iworm_acc_B};
unless (defined $index_B) {
print STDERR "WARNING, no index for iworm acc: $iworm_acc_B\n";
next;
}
print join("\t", $iworm_acc_A, $index_A, $iworm_acc_B, $index_B, $paired_iworm_contigs{$pairing}) . "\n";
}
close $SAM_OFH;
exit(0);
}
####
sub examine_frags {
my ($end_to_iworm_href, $paired_iworm_contigs_href) = @_;
if (exists ($end_to_iworm_href->{LR})) {
my @LR_read_names = keys %{$end_to_iworm_href->{LR}};
for (my $i = 0; $i < $#LR_read_names; $i++) {
for (my $j = $i + 1; $j <= $#LR_read_names; $j++) {
my $pair = join("\t", sort ($LR_read_names[$i], $LR_read_names[$j]) );
$paired_iworm_contigs_href->{$pair}++;
#print STDERR "-got LR pair: $pair\n";
}
}
return;
}
## handle the paired-end reads
my @ends = keys %$end_to_iworm_href;
unless (scalar @ends == 2) {
## no pairs
return;
}
my @iworm_left = keys %{$end_to_iworm_href->{1}};
my @iworm_right = keys %{$end_to_iworm_href->{2}};
if (scalar(@iworm_left) == 1 && scalar(@iworm_right) == 1
&&
$iworm_left[0] ne $iworm_right[0]) {
## simplest case, pairs each aligning to different contigs.
my $i_left = $iworm_left[0];
my $i_right = $iworm_right[0];
my $pair = join("\t", sort ($i_left, $i_right));
#print STDERR "Got pairing: $pair\n";
$paired_iworm_contigs_href->{$pair}++;
}
else {
# examine split reads
&check_for_split_reads($end_to_iworm_href->{1}, $paired_iworm_contigs_href);
&check_for_split_reads($end_to_iworm_href->{2}, $paired_iworm_contigs_href);
}
## output all alignments for these reads.
foreach my $sam_line (values %{$end_to_iworm_href->{1}}, values %{$end_to_iworm_href->{2}}) {
print $SAM_OFH $sam_line;
}
return;
}
####
sub check_for_split_reads {
my ($read_mappings_href, $paired_iworm_contigs_href) = @_;
my @iworm_names = keys %$read_mappings_href;
if (scalar(@iworm_names) < 2) {
# nothing to do here.
return;
}
## see if we have a read where different parts of the read are aligning to different iworm contigs.
for (my $i = 0; $i < $#iworm_names; $i++) {
my $iworm_name_i = $iworm_names[$i];
my $align_i = new SAM_entry($read_mappings_href->{$iworm_name_i});
for (my $j = $i + 1; $j <= $#iworm_names; $j++) {
my $iworm_name_j = $iworm_names[$j];
my $align_j = new SAM_entry($read_mappings_href->{$iworm_name_j});
if (&overlap_not_involving_containment($align_i, $align_j)) {
## store pair
my $pair = join("\t", sort($iworm_name_i, $iworm_name_j));
$paired_iworm_contigs_href->{$pair}++;
}
}
}
return;
}
####
sub overlap_not_involving_containment {
my ($align_i, $align_j) = @_;
my ($read_i_lend, $read_i_rend) = $align_i->get_read_span();
my ($read_j_lend, $read_j_rend) = $align_j->get_read_span();
#print join("\t", "$read_i_lend-$read_i_rend", "$read_j_lend-$read_j_rend") . "\n";
if ( ($read_i_lend <= $read_j_lend && $read_i_rend >= $read_j_rend) # i encapsulates j
||
($read_j_lend <= $read_i_lend && $read_j_rend >= $read_i_rend) # j encapsulates i
) {
## containment
#print "\t* containment\n";
return(0);
}
elsif ( $read_i_lend < $read_j_rend && $read_i_rend > $read_j_lend) {
#print "\t OVERLAP\n";
return(1);
}
else {
#print "\tok\n";
return(1);
}
}
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