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#!/usr/bin/env perl
#
# Copyright 2011-2015, Julian Catchen <jcatchen@illinois.edu>
#
# This file is part of Stacks.
#
# Stacks is free software: you can redistribute it and/or modify
# it under the terms of the GNU General Public License as published by
# the Free Software Foundation, either version 3 of the License, or
# (at your option) any later version.
#
# Stacks is distributed in the hope that it will be useful,
# but WITHOUT ANY WARRANTY; without even the implied warranty of
# MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the
# GNU General Public License for more details.
#
# You should have received a copy of the GNU General Public License
# along with Stacks. If not, see <http://www.gnu.org/licenses/>.
#
#
# Sort paired-end sequences according to the stacks the non-paired-end
# was found in.
#
# By Julian Catchen <jcatchen@illinois.edu>
#
use strict;
use constant stacks_version => "_VERSION_";
use constant true => 1;
use constant false => 0;
my $debug = 0;
my $white_list = "";
my $cat_white_list = "";
my $in_path = "";
my $out_path = "";
my $samp_path = "";
my $out_type = "fasta";
my $in_type = "fastq";
my $gzipped = false;
parse_command_line();
my (@files, %matches, %stacks, %reads, %marker_wl);
build_file_list(\@files);
my ($file, $num_files, $i, $key);
if (length($cat_white_list) > 0) {
load_white_list($cat_white_list, \%marker_wl);
print STDERR "Loaded ", scalar(keys %marker_wl), " catalog IDs from '$cat_white_list'\n";
}
$num_files = scalar(@files);
$i = 1;
foreach $file (@files) {
printf(STDERR "Loading catalog matches, file % 2s of % 2s [%s]\n", $i, $num_files, $file->{'prefix'});
#
# Load the sstacks file, listing matches between the stacks and the catalog
#
load_matches($in_path, $file, \%matches, \%marker_wl);
$i++;
}
#
# Determine which catalog loci have more than a single match from each sample and blacklist them.
#
print STDERR "Identifying catalog loci that have more than one match from a single sample...";
my %multiple_matches;
check_mult_catalog_matches(\%matches, \%multiple_matches);
print STDERR
"done\n",
" These loci will be excluded when collating paired-end reads;\n",
" A list of them has been recorded: $out_path/sort_read_pairs.log\n",
scalar(keys %matches), " total catalog loci; ", scalar(keys %multiple_matches),
" will be excluded, processing ", scalar(keys %matches) - scalar(keys %multiple_matches), " loci.\n";
#
# Check if files already exist, if so, exit to prevent adding data to existing files.
#
my ($cat_id, $path);
foreach $cat_id (keys %matches) {
#
# Check that this catalog ID only has a single match from each sample.
#
next if (defined($multiple_matches{$cat_id}));
$path = $out_path . "/" . $cat_id;
$path .= $out_type eq "fasta" ? ".fa" : ".fq";
if (-e $path) {
die("Error: output files already exist. This program will append data to files if\n" .
"they already exist. Please delete these files and re-execute sort_read_pairs.pl.\n");
}
}
$i = 1;
foreach $file (@files) {
printf(STDERR "Processing file % 2s of % 2s [%s]\n", $i, $num_files, $file->{'prefix'});
#
# Load the ustacks tag file for each sample, containing the read-to-stack mappings
#
$stacks{$file->{'prefix'}} = {};
print STDERR " Loading tag file...";
load_stacks($in_path, $file, $stacks{$file->{'prefix'}});
print STDERR "done.\n";
#
# Map the read-pairs to the stack/catalog match they correspond to.
#
$reads{$file->{'prefix'}} = {};
print STDERR " Loading sample file...";
$in_type eq "fastq" ?
process_fastq_read_pairs($samp_path, $file, \%stacks, $reads{$file->{'prefix'}}) :
process_fasta_read_pairs($samp_path, $file, \%stacks, $reads{$file->{'prefix'}});
print STDERR "done.\n";
print STDERR " Printing results...";
print_results($out_path, \%matches, \%stacks, \%reads, \%multiple_matches);
print STDERR "done.\n";
#
# Clean up memory usage.
#
print STDERR " Clearing memory...";
undef(%{$stacks{$file->{'prefix'}}});
undef(%{$reads{$file->{'prefix'}}});
print STDERR " done.\n";
$i++;
}
sub load_matches {
my ($in_path, $in_file, $matches, $marker_wl) = @_;
my ($file, $in_fh, $line, @parts, $key);
if ($gzipped == true) {
$file = $in_path . "/" . $in_file->{'prefix'} . $in_file->{'suffix'} . ".matches.tsv.gz";
open($in_fh, "gunzip -c $file |") or die("Unable to open catalog matches file '$file', $!\n");
} else {
$file = $in_path . "/" . $in_file->{'prefix'} . $in_file->{'suffix'} . ".matches.tsv";
open($in_fh, "<$file") or die("Unable to open catalog matches file '$file', $!\n");
}
while ($line = <$in_fh>) {
chomp $line;
@parts = split(/\t/, $line);
if (length($cat_white_list) > 0) {
next if (!defined($marker_wl->{$parts[2]}));
}
if (!defined($matches->{$parts[2]})) {
$matches->{$parts[2]} = {};
}
#
# Index by catalog_ID -> sample_ID|stack_ID
#
$key = $in_file->{'prefix'} . "|" . $parts[4];
$matches->{$parts[2]}->{$key}++;
}
close($in_fh);
}
sub load_stacks {
my ($in_path, $in_file, $stacks) = @_;
my ($file, $in_fh, $line, @parts);
if ($gzipped == true) {
$file = $in_path . "/" . $in_file->{'prefix'} . $in_file->{'suffix'} . ".tags.tsv.gz";
open($in_fh, "gunzip -c $file |") or die("Unable to open '$file', $!\n");
} else {
$file = $in_path . "/" . $in_file->{'prefix'} . $in_file->{'suffix'} . ".tags.tsv";
open($in_fh, "<$file") or die("Unable to open '$file', $!\n");
}
while ($line = <$in_fh>) {
chomp $line;
@parts = split(/\t/, $line);
next if ($parts[6] eq "consensus" || $parts[6] eq "model");
#
# Index by sequence ID -> stack ID
#
$stacks->{substr($parts[8], 0, -2)} = $parts[2];
}
close($in_fh);
}
sub process_fastq_read_pairs {
my ($in_path, $in_file, $stacks, $reads) = @_;
my ($file, $in_fh, $line, $seq, $qual, $key, $read_id);
if ($in_file->{'suffix'} eq ".1") {
if ($gzipped == true) {
$file = $in_path . "/" . $in_file->{'prefix'} . ".2.fq.gz";
open($in_fh, "gunzip -c $file |") or die("Unable to open paired-end input file '$file'\n");
} else {
$file = $in_path . "/" . $in_file->{'prefix'} . ".2.fq";
open($in_fh, "<$file") or die("Unable to open paired-end input file '$file'\n");
}
} else {
if ($gzipped == true) {
$file = $in_path . "/" . $in_file->{'prefix'} . ".fq_2.gz";
open($in_fh, "gunzip -c $file |") or die("Unable to open paired-end input file '$file'\n");
} else {
$file = $in_path . "/" . $in_file->{'prefix'} . ".fq_2";
open($in_fh, "<$file") or die("Unable to open paired-end input file '$file'\n");
}
}
while ($line = <$in_fh>) {
next if (substr($line, 0, 1) ne "@");
chomp $line;
$read_id = substr($line, 1, -2);
$seq = <$in_fh>;
chomp $seq;
#
# Read the repeated ID and the quality scores.
#
<$in_fh>;
$qual = <$in_fh>;
chomp $qual;
$key = $stacks->{$in_file->{'prefix'}}->{$read_id};
next if (!defined($key));
if (!defined($reads->{$key})) {
$reads->{$key} = [];
}
push(@{$reads->{$key}}, {'id' => $read_id, 'seq' => $seq, 'qual' => $qual});
}
}
sub process_fasta_read_pairs {
my ($in_path, $in_file, $stacks, $reads) = @_;
my ($file, $in_fh, $line, $seq, $qual, $key, $read_id);
if ($in_file->{'suffix'} eq ".1") {
if ($gzipped == true) {
$file = $in_path . "/" . $in_file->{'prefix'} . ".2.fa.gz";
open($in_fh, "gunzip -c $file |") or die("Unable to open paired-end input file '$file'\n");
} else {
$file = $in_path . "/" . $in_file->{'prefix'} . ".2.fa";
open($in_fh, "<$file") or die("Unable to open paired-end input file '$file'\n");
}
} else {
if ($gzipped == true) {
$file = $in_path . "/" . $in_file->{'prefix'} . ".fa_2.gz";
open($in_fh, "gunzip -c $file |") or die("Unable to open paired-end input file '$file'\n");
} else {
$file = $in_path . "/" . $in_file->{'prefix'} . ".fa_2";
open($in_fh, "<$file") or die("Unable to open paired-end input file '$file'\n");
}
}
while ($line = <$in_fh>) {
next if (substr($line, 0, 1) ne ">");
chomp $line;
$read_id = substr($line, 1, -2);
$seq = <$in_fh>;
chomp $seq;
$key = $stacks->{$in_file->{'prefix'}}->{$read_id};
next if (!defined($key));
if (!defined($reads->{$key})) {
$reads->{$key} = [];
}
push(@{$reads->{$key}}, {'id' => $read_id, 'seq' => $seq, 'qual' => ""});
}
}
sub print_results {
my ($out_path, $matches, $stacks, $reads, $multiple_matches) = @_;
my ($path, $cat_id, $sample, $stack_id, $read, $out_fh, $i, @keys, $count, $key, $mult_hits);
#
# If a catalog ID matches stacks from multiple samples, print them out together.
#
foreach $cat_id (keys %{$matches}) {
#
# Check that this catalog ID only has a single match from each sample.
#
next if (defined($multiple_matches->{$cat_id}));
$path = $out_path . "/" . $cat_id;
$path .= $out_type eq "fasta" ? ".fa" : ".fq";
open($out_fh, ">>$path") or die("Unable to open $path; '$!'\n");
foreach $key (keys %{$matches->{$cat_id}}) {
($sample, $stack_id) = split(/\|/, $key);
foreach $read (@{$reads->{$sample}->{$stack_id}}) {
if ($out_type eq "fasta") {
print $out_fh
">", $cat_id, "|", $sample, "|", $stack_id, "|", $read->{'id'}, "\n",
$read->{'seq'}, "\n";
} else {
print $out_fh
"@", $cat_id, "|", $sample, "|", $stack_id, "|", $read->{'id'}, "\n",
$read->{'seq'}, "\n",
"+\n",
$read->{'qual'}, "\n";
}
}
}
close($out_fh);
}
}
sub check_mult_catalog_matches {
my ($matches, $multiple_matches) = @_;
my ($fh, $key, $sample, $stack_id);
#
# Find catalog loci that have more than a single match from one or more samples
# and log those loci that will be excluded.
#
open($fh, ">$out_path/sort_read_pairs.log") or die("Unable to open log file, $!\n");
print $fh
"# The catalog loci listed below have more than a single match from one or more individuals, indicating undermerged or repetitive loci.\n",
"# CatalogLocus <tab> Sample1:Locus1,Locus2;Sample2:Locus1,Locus2\n";
foreach $cat_id (keys %{$matches}) {
my %samples;
foreach $key (keys %{$matches->{$cat_id}}) {
($sample, $stack_id) = split(/\|/, $key);
push(@{$samples{$sample}}, $stack_id);
}
my $mult_hits = 0;
my $str = "";
foreach $sample (keys %samples) {
if (scalar(@{$samples{$sample}}) > 1) {
$mult_hits++;
$str .= $sample . ":" . join(",", @{$samples{$sample}}) . "; ";
}
}
if ($mult_hits > 0) {
print $fh $cat_id, "\t", substr($str, 0, -1), "\n";
$multiple_matches{$cat_id}++;
}
}
close($fh);
}
sub count_reads {
my ($catalog, $reads) = @_;
my ($count, $key, $sample, $stack_id);
$count = 0;
foreach $key (keys %{$catalog}) {
($sample, $stack_id) = split(/\|/, $key);
if (defined($reads->{$sample}->{$stack_id})) {
$count += scalar(@{$reads->{$sample}->{$stack_id}});
}
}
return $count;
}
sub build_file_list {
my ($files) = @_;
my (@ls, $line, $file, $prefix, $suffix);
# Load a white list of files to process if it is supplied.
my %wl;
if (length($white_list) > 0) {
load_white_list($white_list, \%wl);
print STDERR "Loaded ", scalar(keys %wl), " filenames from '$white_list'\n";
}
@ls = glob("$in_path/*.tags.tsv");
if (scalar @ls == 0) {
@ls = glob("$in_path/*.tags.tsv.gz");
$gzipped = true if (scalar @ls > 0);
}
foreach $line (@ls) {
chomp $line;
next if (length($line) == 0);
next if ($line =~ /batch_\d+\.catalog/);
($file) = ($line =~ /$in_path\/(.+)\.tags\.tsv\.?g?z?$/);
if (length($white_list) > 0) {
next if (!defined($wl{$file}));
}
if ($file =~ /\.1$/) {
($prefix, $suffix) = ($file =~ /^(.+)(\.1)$/);
} else {
$prefix = $file;
$suffix = "";
}
push(@{$files}, {'prefix' => $prefix, 'suffix' => $suffix});
}
}
sub load_white_list {
my ($list, $wl) = @_;
open(WHITE, "<" . $list)
or die("Unable to open white list file '$white_list': $!\n");
my $line = "";
while ($line = <WHITE>) {
chomp $line;
next if (length($line) == 0);
next if ($line =~ /^\s*#/);
$wl->{$line}++;
}
close(WHITE);
}
sub parse_command_line {
while (@ARGV) {
$_ = shift @ARGV;
if ($_ =~ /^-p$/) { $in_path = shift @ARGV; }
elsif ($_ =~ /^-o$/) { $out_path = shift @ARGV; }
elsif ($_ =~ /^-s$/) { $samp_path = shift @ARGV; }
elsif ($_ =~ /^-t$/) { $out_type = shift @ARGV; }
elsif ($_ =~ /^-i$/) { $in_type = shift @ARGV; }
elsif ($_ =~ /^-W$/) { $white_list = shift @ARGV; }
elsif ($_ =~ /^-w$/) { $cat_white_list = shift @ARGV; }
elsif ($_ =~ /^-d$/) { $debug++; }
elsif ($_ =~ /^-v$/) { version(); exit(); }
elsif ($_ =~ /^-h$/) { usage(); }
else {
print STDERR "Unknown command line option: '$_'\n";
usage();
}
}
if ($out_type ne "fasta" && $out_type ne "fastq") {
print STDERR "Output type must be either 'fasta' or 'fastq'.\n";
usage();
}
if ($in_type ne "fasta" && $in_type ne "fastq") {
print STDERR "Input type must be either 'fasta' or 'fastq'.\n";
usage();
}
if (length($in_path) == 0) {
print STDERR "You must specify a path to the Stacks output files.\n";
usage();
}
if (length($out_path) == 0) {
print STDERR "You must specify a path to write the collated output files.\n";
usage();
}
if (length($samp_path) == 0) {
print STDERR "You must specify a path to the paired-end reads.\n";
usage();
}
$in_path = substr($in_path, 0, -1) if (substr($in_path, -1) eq "/");
$out_path = substr($out_path, 0, -1) if (substr($out_path, -1) eq "/");
$samp_path = substr($samp_path, 0, -1) if (substr($samp_path, -1) eq "/");
}
sub version {
print STDERR "sort_read_pairs.pl ", stacks_version, "\n";
}
sub usage {
version();
print STDERR <<EOQ;
sort_read_pairs.pl -p path -s path -o path [-t type] [-W white_list] [-w white_list] [-d] [-h]
p: path to the Stacks output files.
s: path to paired-end sample files.
o: path to output the collated FASTA files.
i: input type, either 'fasta' or 'fastq' (default).
t: output type, either 'fasta' (default) or 'fastq'.
W: a white list of files to process in the input path.
w: a white list of catalog IDs to include.
h: display this help message.
d: turn on debug output.
EOQ
exit(0);
}
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