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|
#!/usr/bin/perl -w
##
# mapability.pl
#
# Calculate mapability of each reference position assuming reference is
# bisulfite treated.
#
use strict;
use warnings;
use Getopt::Long;
my $fa = "";
my $win = 50;
my $freq = 1;
my $bowtie = "";
my $bowtie_arg = "";
my $pol = "-v 3";
my $fwidx = "";
my $rcidx = "";
my $btargs = "-t --norc -S --sam-nohead -M 1 --mm";
my $debug = 0;
if(defined($ENV{BOWTIE_HOME})) {
$bowtie = "$ENV{BOWTIE_HOME}/bowtie";
unless(-x $bowtie) { $bowtie = "" };
}
if($bowtie eq "") {
$bowtie = `which bowtie 2>/dev/null`;
chomp($bowtie);
unless(-x $bowtie) { $bowtie = "" };
}
$bowtie = "./bowtie" if ($bowtie eq "" && -x "./bowtie");
GetOptions(
"fasta=s" => \$fa,
"window=i" => \$win,
"frequency=i" => \$freq,
"bowtie=s" => \$bowtie_arg,
"policy=s" => \$pol,
"fwidx=s" => \$fwidx,
"rcidx=s" => \$rcidx,
"debug" => \$debug
) || die;
print STDERR "Bowtie: found: $bowtie; given: $bowtie_arg\n";
print STDERR "Input fasta: $fa\n";
print STDERR "FW index: $fwidx\n";
print STDERR "RC index: $rcidx\n";
print STDERR "Alignment policy: $pol\n";
print STDERR "Window size: $win\n";
print STDERR "Frequency: $freq\n";
$fa ne "" || die "Must specify -fasta\n";
$fwidx ne "" || die "Must specify -fwidx\n";
$rcidx ne "" || die "Must specify -rcidx\n";
-f "$fwidx.1.ebwt" || die "Could not find -fwidx index file $fwidx.1.ebwt\n";
-f "$rcidx.1.ebwt" || die "Could not find -rcidx index file $rcidx.1.ebwt\n";
$bowtie = $bowtie_arg if $bowtie_arg ne "";
unless(-x $bowtie) {
# No bowtie? die
if($bowtie_arg ne "") {
die "Specified -bowtie, \"$bowtie\" doesn't exist or isn't executable\n";
} else {
die "bowtie couldn't be found in BOWTIE_HOME, PATH, or current directory; please specify -bowtie\n";
}
}
my $running = 0;
my $name = ""; # name of sequence currently being processed
my %lens = ();
my @names = ();
my $totlen = 0;
##
# Read lengths of all the entries in all the input fasta files.
#
sub readLens($) {
my $ins = shift;
my @is = split(/[,]/, $ins);
for my $i (@is) {
open IN, "$i" || die "Could not open $i\n";
my $name = "";
while(<IN>) {
chomp;
if(substr($_, 0, 1) eq '>') {
next if /\?[0-9]*$/; # Skip >?50000 lines
$name = $_;
$name = substr($name, 1); # Chop off >
if($name =~ /^FW:/ || $name =~ /^RC:/) {
$name = substr($name, 3); # Chop off FW:/RC:
}
my @ns = split(/\s+/, $name);
$name = $ns[0]; # Get short name
push @names, $name;
print STDERR "Saw name $name\n";
} else {
$name ne "" || die;
$lens{$name} += length($_); # Update length
$totlen += length($_);
}
}
close(IN);
}
}
print STDERR "Reading fasta lengths\n";
readLens($fa);
print STDERR " read ".scalar(keys %lens)." sequences with total length $totlen\n";
my @last;
for(my $i = 0; $i < $win; $i++) { push @last, 0 };
sub clearLast {
for(my $i = 0; $i < $win; $i++) { $last[$i] = 0 };
}
print STDERR "Opening bowtie pipes\n";
my $fwcmd = "$bowtie -F $win,$freq $btargs $pol $fwidx $fa";
my $rccmd = "$bowtie -F $win,$freq $btargs $pol $rcidx $fa";
print STDERR "Forward command: $fwcmd\n";
print STDERR "Reverse comp command: $rccmd\n";
open BTFW, "$fwcmd |" || die "Couldn't open pipe '$fwcmd |'\n";
open BTRC, "$rccmd |" || die "Couldn't open pipe '$rccmd |'\n";
# 10_554 4 * 0 0 * * 0 0 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII XM:i:0
# 10_555 4 * 0 0 * * 0 0 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII XM:i:0
# 10_556 4 * 0 0 * * 0 0 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII XM:i:0
# 10_557 4 * 0 0 * * 0 0 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII XM:i:0
# 10_558 4 * 0 0 * * 0 0 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII XM:i:0
# 10_559 4 * 0 0 * * 0 0 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII XM:i:0
print STDERR "Reading...\n";
my $ln = 0;
my $cur = 0;
my $lastc = "\n";
while(1) {
my $fwl = <BTFW>;
my $rcl = <BTRC>;
last unless defined($fwl) && defined($rcl);
$ln++;
my @fws = split(/\t/, $fwl);
my @fws1 = split(/_/, $fws[0]);
my @rcs = split(/\t/, $rcl);
my @rcs1 = split(/_/, $rcs[0]);
my $cname = join("_", @fws1[0..$#fws1-1]);
$cname eq join("_", @fws1[0..$#fws1-1]) || die "Name mismatch on line $ln:\n$fwl\n$rcl\n";
my $off = $fws1[-1];
$off eq $rcs1[-1] || die "Offset mismatch on line $ln:\n$fwl\n$rcl\n";
# If a read aligns, XM:i is not printed
# If a read fails to align, XM:i:0 is printed
# If a read aligns multiple places, XM:i:N is printed where N>0
my $fwxm0 = $fws[-1] =~ /XM:i:0/;
my $rcxm0 = $rcs[-1] =~ /XM:i:0/;
my $fwxm = $fws[-1] =~ /XM:i:/;
my $rcxm = $rcs[-1] =~ /XM:i:/;
# For mapable to be true, we need the read to have failed to align
# in one index and aligned uniquely in the other
my $mapable = (($fwxm0 && !$rcxm) || ($rcxm0 && !$fwxm)) ? 1 : 0;
$cname =~ s/\s.*//; # trim everything after first whitespace to get short name
if($cname =~ /^FW:/ || $cname =~ /^RC:/) {
$cname = substr($cname, 3);
}
if($name ne $cname) {
if($name ne "") {
# Flush remaining characters from previous name
defined($lens{$name}) || die;
$cur == $lens{$name} - $win + 1 || die "name is $name, cur is $cur, len is $lens{$name}, win is $win\n";
for(; $cur < $lens{$name}; $cur++) {
$running -= $last[$cur % $win];
$last[$cur % $win] = 0;
$lastc = chr($running + 64);
print $lastc;
if((($cur+1) % 60) == 0) {
$lastc = "\n";
print $lastc;
}
}
}
$name = $cname;
defined($lens{$name}) || die "No such name as \"$name\"\n";
print "\n" unless $lastc eq "\n";
print ">$name\n";
$lastc = "\n";
$cur = 0;
$running = 0;
clearLast();
}
$running -= $last[$cur % $win];
$last[$cur % $win] = $mapable;
$running += $last[$cur % $win];
$running <= $win || die "running counter $running got higher than window size $win\n";
$lastc = chr($running + 64);
print $lastc;
if((($cur+1) % 60) == 0) {
$lastc = "\n";
print $lastc;
}
$cur++;
}
defined($lens{$name}) || die;
for(; $cur < $lens{$name}; $cur++) {
$running -= $last[$cur % $win];
$last[$cur % $win] = 0;
$lastc = chr($running + 64);
print $lastc;
if((($cur+1) % 60) == 0) {
$lastc = "\n";
print $lastc;
}
}
close(BTFW);
$? == 0 || die "Bad exitlevel from forward bowtie: $?\n";
close(BTRC);
$? == 0 || die "Bad exitlevel from reverse-comp bowtie: $?\n";
|
