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|
#!/usr/bin/perl -w
#
# Verifies that Bowtie's --better and --best alignment modes really
# produce alignments that are the best possible in terms of score.
#
# Run this from the Bowtie directory.
#
# E.g.: perl ../scripts/best_verify.pl -v 0
# perl ../scripts/best_verify.pl -v 0 e_coli genomes/NC_008253.fna reads/e_coli_1000.fq
#
use warnings;
use strict;
use Getopt::Long;
my %options=();
my $varg;
my $narg;
my $g = undef;
my $l = undef;
my $e = undef;
my $C = undef;
my $debug = 0;
my $round = "";
my $best = undef;
my $nomaqround = 0;
my $result = GetOptions(
"v=i" => \$varg,
"n=i" => \$narg,
"l=i" => \$l,
"e=i" => \$e,
"d" => \$debug,
"g" => \$g,
"best"=> \$best,
"C" => \$C,
"nomaqround" => \$nomaqround) || die "Bad options";
my $match_mode = "-n 2";
$match_mode = " -v " . $varg if defined($varg);
$match_mode = " -n " . $narg if defined($narg);
$match_mode .= " -l $l " if defined($l);
$match_mode .= " -e $e " if defined($e);
$match_mode .= " -g " if defined($g);
$match_mode .= " -C " if defined($C);
$match_mode .= " --cost ";
$round = "--nomaqround" if $nomaqround;
my $seedLen = 28;
$seedLen = $l if defined($l);
print "Maq-like rounding is: ".($nomaqround ? "off" : "on") . "\n";
print "Using match mode: $match_mode\n";
if(defined($C)) {
print "Aborting with exitlevel 0 because colorspace alignment was selected\n";
exit 0;
}
my $bowtie_dir = ".";
my $bowtie_exe = "bowtie";
$bowtie_exe .= " --debug" if $debug;
my $index = "e_coli";
$index = $ARGV[0] if defined($ARGV[0]);
my $reads = "reads/e_coli_1000.fq";
$reads = $ARGV[1] if defined($ARGV[1]);
if($reads =~ /\.fa$/) {
$reads = " -f $reads ";
} elsif($reads =~ /\.raw$/) {
$reads = " -r $reads ";
} elsif($reads =~ /\.integer.fq$/) {
$reads = " -q --integer-quals $reads ";
} elsif($reads =~ /\.fq$/) {
$reads = " -q $reads ";
} else {
$reads = " -c $reads ";
}
my $vmode = ($match_mode =~ /[-]v/);
system("make -C $bowtie_dir $bowtie_exe") == 0 || die;
# Run Bowtie to get best alignments
my $bowtie_best_cmd = "$bowtie_dir/$bowtie_exe -y $round $match_mode --best --refidx $index $reads";
# Run Bowtie to get all alignments
my $bowtie_all_cmd = "$bowtie_dir/$bowtie_exe -y $round $match_mode -a --refidx $index $reads";
print "$bowtie_best_cmd\n";
open BOWTIE_BEST, "$bowtie_best_cmd |";
my %nameToBestScore = ();
my %nameToBestAlignment = ();
my $bestAls = 0;
while(<BOWTIE_BEST>) {
next if /^Reported/;
chomp;
my $line = $_;
my @ls = split(/[\t]/, $line);
my $btcost = $ls[-1];
$#ls >= 5 || die "Alignment not formatted correctly: $line";
my $name = $ls[0];
defined($nameToBestAlignment{$name}) && die "Read with name $name appeared more than once in best-hit output";
defined($nameToBestScore{$name}) && die "Read with name $name appeared more than once in best-hit output";
my $len = length($ls[4]);
my $quals = $ls[5];
my $mmstr = "";
$mmstr = $ls[7] if defined($ls[7]);
my $cost = 0;
my $fw = $ls[1] eq "+";
$seedLen = $len if $vmode;
if($mmstr ne "-") {
my @mms = split(/,/, $mmstr);
for my $mm (@mms) {
my @mmss = split(/:/, $mm);
my $mmoff = int($mmss[0]);
if($mmoff < $seedLen) {
$cost += (1 << 14);
}
if(!$fw) {
$mmoff = $len - $mmoff - 1;
}
my $q = substr($quals, $mmoff, 1);
$q = int(ord($q) - 33);
if(!$nomaqround) {
$q = int(int($q + 5) / 10);
$q = 3 if $q > 3;
$q *= 10;
}
my $qcost += $q;
$cost += $qcost;
}
}
print "$line: $cost\n";
$cost == $btcost || die "script-calculated cost $cost doesn't match bowtie-calculated cost $btcost\n";
$nameToBestAlignment{$name} = $line;
$nameToBestScore{$name} = $cost;
$bestAls++;
}
close(BOWTIE_BEST);
if($? != 0) {
die "bowtie --best process failed with exitlevel $?\n";
}
print "$bowtie_all_cmd\n";
open BOWTIE_ALL, "$bowtie_all_cmd |";
my $allAls = 0;
while(<BOWTIE_ALL>) {
next if /^Reported/;
chomp;
my $line = $_;
my @ls = split(/[\t]/, $line);
my $btcost = $ls[-1];
$#ls >= 5 || die "Alignment not formatted correctly: $line";
my $name = $ls[0];
my $len = length($ls[4]);
my $quals = $ls[5];
my $mmstr = "";
$mmstr = $ls[7] if defined($ls[7]);
my $cost = 0;
my $fw = $ls[1] eq "+";
$seedLen = $len if $vmode;
if($mmstr ne "-") {
my @mms = split(/,/, $mmstr);
for my $mm (@mms) {
my @mmss = split(/:/, $mm);
my $mmoff = int($mmss[0]);
if($mmoff < $seedLen) {
$cost += (1 << 14);
}
if(!$fw) {
$mmoff = $len - $mmoff - 1;
}
my $q = substr($quals, $mmoff, 1);
$q = int(ord($q) - 33);
if(!$nomaqround) {
$q = int(int($q + 5) / 10);
$q = 3 if $q > 3;
$q *= 10;
}
my $qcost += $q;
$cost += $qcost;
}
}
print "$line: $cost\n";
$cost == $btcost || die "script-calculated cost $cost doesn't match bowtie-calculated cost $btcost\n";
defined($nameToBestAlignment{$name}) ||
die "Read with alignment:\n$line\nhas no corresponding alignment in best-hit mode\n";
int($cost) >= int($nameToBestScore{$name}) ||
die "Alignment:\n$line\n$cost\nis better than:\n$nameToBestAlignment{$name}\n$nameToBestScore{$name}\n";
$allAls++;
}
close(BOWTIE_ALL);
if($? != 0) {
die "bowtie -a process failed with exitlevel $?\n";
}
print "Checked $bestAls best-alignments against $allAls all-alignments\n";
print "PASSED\n";
|
