use warnings;
use Getopt::Std;
-my $version = '0.2.3';
+my $version = '0.3.3';
&usage if (@ARGV < 1);
my $command = shift(@ARGV);
-my %func = (snpFilter=>\&snpFilter, indelFilter=>\&indelFilter, showALEN=>\&showALEN, pileup2fq=>\&pileup2fq);
+my %func = (showALEN=>\&showALEN, pileup2fq=>\&pileup2fq, varFilter=>\&varFilter, plp2vcf=>\&plp2vcf,
+ unique=>\&unique, uniqcmp=>\&uniqcmp, sra2hdr=>\&sra2hdr, sam2fq=>\&sam2fq);
die("Unknown command \"$command\".\n") if (!defined($func{$command}));
&{$func{$command}};
die(qq/Usage: samtools.pl showALEN <in.sam>\n/) if (@ARGV == 0 && -t STDIN);
while (<>) {
my @t = split;
+ next if (/^\@/ || @t < 11);
my $l = 0;
$_ = $t[5];
- s/(\d+)[SMI]/$l+=$1/eg;
+ s/(\d+)[MI]/$l+=$1/eg;
print join("\t", @t[0..5]), "\t$l\t", join("\t", @t[6..$#t]), "\n";
}
}
#
-# indelFilter
+# varFilter
#
-sub indelFilter {
- my %opts = (D=>100, m=>10, r=>undef, s=>100);
- getopts('D:m:rs:', \%opts); # -s for scaling factor in score calculation
+#
+# Filtration code:
+#
+# d low depth
+# D high depth
+# W too many SNPs in a window (SNP only)
+# G close to a high-quality indel (SNP only)
+# Q low RMS mapping quality (SNP only)
+# g close to another indel with higher quality (indel only)
+# s low SNP quality (SNP only)
+# i low indel quality (indel only)
+sub varFilter {
+ my %opts = (d=>3, D=>100, l=>30, Q=>25, q=>10, G=>25, s=>100, w=>10, W=>10, N=>2, p=>undef, S=>'', i=>'');
+ getopts('pq:d:D:l:Q:w:W:N:G:S:i:', \%opts);
die(qq/
-Usage: samtools.pl indelFilter [options] <in.indel>\n
-Options: -D INT maximum read depth [$opts{D}]
- -m INT minimum distance between two adjacent indels [$opts{m}]
+Usage: samtools.pl varFilter [options] <in.cns-pileup>
+
+Options: -Q INT minimum RMS mapping quality for SNPs [$opts{Q}]
+ -q INT minimum RMS mapping quality for gaps [$opts{q}]
+ -d INT minimum read depth [$opts{d}]
+ -D INT maximum read depth [$opts{D}]
+ -S INT minimum SNP quality [$opts{S}]
+ -i INT minimum indel quality [$opts{i}]
+
+ -G INT min indel score for nearby SNP filtering [$opts{G}]
+ -w INT SNP within INT bp around a gap to be filtered [$opts{w}]
+
+ -W INT window size for filtering dense SNPs [$opts{W}]
+ -N INT max number of SNPs in a window [$opts{N}]
+
+ -l INT window size for filtering adjacent gaps [$opts{l}]
+
+ -p print filtered variants
\n/) if (@ARGV == 0 && -t STDIN);
- my (@arr1, @arr2);
- my ($curr, $last) = (\@arr1, \@arr2);
- my $is_ref = defined($opts{r})? 1 : 0;
+ # calculate the window size
+ my ($ol, $ow, $oW) = ($opts{l}, $opts{w}, $opts{W});
+ my $max_dist = $ol > $ow? $ol : $ow;
+ $max_dist = $oW if ($max_dist < $oW);
+ # the core loop
+ my @staging; # (indel_filtering_score, flt_tag)
while (<>) {
my @t = split;
- next if ($t[2] ne '*');
- if (!$is_ref) {
- next if ($t[3] eq '*/*');
- next if ($t[5] == 0);
- }
- next if ($t[7] > $opts{D});
- # calculate indel score
- my $score = $t[5];
- $score += $opts{s} * $t[10] if ($t[8] ne '*');
- $score += $opts{s} * $t[11] if ($t[9] ne '*');
- @$curr = ($t[0], $t[1], $score, $_);
- my $do_swap = 1;
- if (defined $last->[0]) {
- if ($curr->[0] eq $last->[0] && $last->[1] + $opts{m} > $curr->[1]) {
- $do_swap = 0 if ($last->[2] > $curr->[2]);
- } else { # then print
- print $last->[3];
- }
+ next if (uc($t[2]) eq uc($t[3]) || $t[3] eq '*/*'); # skip non-var sites
+ # clear the out-of-range elements
+ while (@staging) {
+ # Still on the same chromosome and the first element's window still affects this position?
+ last if ($staging[0][3] eq $t[0] && $staging[0][4] + $staging[0][2] + $max_dist >= $t[1]);
+ varFilter_aux(shift(@staging), $opts{p}); # calling a function is a bit slower, not much
}
- if ($do_swap) {
- my $tmp = $curr; $curr = $last; $last = $tmp;
+ my ($flt, $score) = (0, -1);
+ # first a simple filter
+ if ($t[7] < $opts{d}) {
+ $flt = 2;
+ } elsif ($t[7] > $opts{D}) {
+ $flt = 3;
}
- }
- print $last->[3] if (defined $last->[0]);
-}
+ if ($t[2] eq '*') { # an indel
+ if ($opts{i} && $opts{i}>$t[5]) { $flt = 8; }
+ }
+ elsif ($opts{S} && $opts{S}>$t[5]) { $flt = 7; } # SNP
-#
-# snpFilter
-#
-
-sub snpFilter {
- my %opts = (f=>'', Q=>40, d=>3, w=>10, D=>0, N=>2, W=>10, q=>20, s=>50);
- getopts('f:s:w:q:Q:d:D:W:N:', \%opts);
- die(qq{
-Usage: samtools.pl snpFilter [options] <cns2snp.snp>
-
-Options: -d INT minimum depth to call a SNP [$opts{d}]
- -D INT maximum depth, 0 to ignore [$opts{D}]
- -Q INT required max mapping quality of the reads covering the SNP [$opts{Q}]
- -q INT minimum SNP quality [$opts{q}]
-
- -f FILE filtered samtools indels [null]
- -s INT minimum samtols indel score [$opts{s}]
- -w INT SNP within INT bp around an indel to be filtered [$opts{w}]
-
- -W INT window size for filtering dense SNPs [$opts{W}]
- -N INT maximum number of SNPs in a window [$opts{N}]
-\n}) unless (@ARGV);
- my (%hash, $fh);
- my $skip = $opts{w};
- $opts{D} = 100000000 if ($opts{D} == 0);
- if ($opts{f}) { # filtered samtools indel
- my $n = 0;
- open($fh, $opts{f}) || die;
- while (<$fh>) {
- my @t = split;
- next if ($t[2] ne '*' || $t[3] eq '*/*' || $t[5] < $opts{s});
- for (my $x = $t[1] - $skip + 1; $x < $t[1] + $skip; ++$x) {
- $hash{$t[0],$x} = 1;
+ # site dependent filters
+ my $len=0;
+ if ($flt == 0) {
+ if ($t[2] eq '*') { # an indel
+ # If deletion, remember the length of the deletion
+ my ($a,$b) = split(m{/},$t[3]);
+ my $alen = length($a) - 1;
+ my $blen = length($b) - 1;
+ if ( $alen>$blen )
+ {
+ if ( substr($a,0,1) eq '-' ) { $len=$alen; }
+ }
+ elsif ( substr($b,0,1) eq '-' ) { $len=$blen; }
+
+ $flt = 1 if ($t[6] < $opts{q});
+ # filtering SNPs
+ if ($t[5] >= $opts{G}) {
+ for my $x (@staging) {
+ # Is it a SNP and is it outside the SNP filter window?
+ next if ($x->[0] >= 0 || $x->[4] + $x->[2] + $ow < $t[1]);
+ $x->[1] = 5 if ($x->[1] == 0);
+ }
+ }
+ # calculate the filtering score (different from indel quality)
+ $score = $t[5];
+ $score += $opts{s} * $t[10] if ($t[8] ne '*');
+ $score += $opts{s} * $t[11] if ($t[9] ne '*');
+ # check the staging list for indel filtering
+ for my $x (@staging) {
+ # Is it a SNP and is it outside the gap filter window
+ next if ($x->[0] < 0 || $x->[4] + $x->[2] + $ol < $t[1]);
+ if ($x->[0] < $score) {
+ $x->[1] = 6;
+ } else {
+ $flt = 6; last;
+ }
+ }
+ } else { # a SNP
+ $flt = 1 if ($t[6] < $opts{Q});
+ # check adjacent SNPs
+ my $k = 1;
+ for my $x (@staging) {
+ ++$k if ($x->[0] < 0 && $x->[4] + $x->[2] + $oW >= $t[1] && ($x->[1] == 0 || $x->[1] == 4 || $x->[1] == 5));
+ }
+ # filtering is necessary
+ if ($k > $opts{N}) {
+ $flt = 4;
+ for my $x (@staging) {
+ $x->[1] = 4 if ($x->[0] < 0 && $x->[4] + $x->[2] + $oW >= $t[1] && $x->[1] == 0);
+ }
+ } else { # then check gap filter
+ for my $x (@staging) {
+ next if ($x->[0] < 0 || $x->[4] + $x->[2] + $ow < $t[1]);
+ if ($x->[0] >= $opts{G}) {
+ $flt = 5; last;
+ }
+ }
+ }
}
}
- close($fh);
+ push(@staging, [$score, $flt, $len, @t]);
}
- my (@last, $last_chr);
- $last_chr = '';
- while (<>) {
- my @t = split;
- next if ($t[2] eq '*' || $hash{$t[0],$t[1]});
- next if ($t[2] eq $t[3]);
- my $is_good = ($t[7] >= $opts{d} && $t[7] <= $opts{D} && $t[6] >= $opts{Q} && $t[5] >= $opts{q})? 1 : 0;
- next unless ($is_good); # drop
- if ($t[0] ne $last_chr) { # a different chr, print
- map { print $_->{L} if ($_->{F}) } @last;
- @last = ();
- $last_chr = $t[0];
- }
- # The following block implemented by Nathans Weeks.
- push(@last, {L => $_, X => $t[1], F => 1}); # Enqueue current SNP
- if ($#last == $opts{N}) { # number of SNPs in queue is N+1
- if ($last[$#last]{X} - $last[0]{X} < $opts{W}) { # if all within window W
- map {$_->{F} = 0} @last; # all SNPs in the window of size W are "bad"
- }
- print STDOUT $last[0]{L} if ($last[0]{F}); # print first SNP if good
- shift @last # dequeue first SNP
- }
+ # output the last few elements in the staging list
+ while (@staging) {
+ varFilter_aux(shift @staging, $opts{p});
+ }
+}
+
+sub varFilter_aux {
+ my ($first, $is_print) = @_;
+ if ($first->[1] == 0) {
+ print join("\t", @$first[3 .. @$first-1]), "\n";
+ } elsif ($is_print) {
+ print STDERR join("\t", substr("UQdDWGgsiX", $first->[1], 1), @$first[3 .. @$first-1]), "\n";
}
- # print the last few lines if applicable
- map { print $_->{L} if ($_->{F}) } @last;
}
+#
+# pileup2fq
+#
+
sub pileup2fq {
- my %opts = (d=>3, D=>255, Q=>25, G=>50, l=>10);
+ my %opts = (d=>3, D=>255, Q=>25, G=>25, l=>10);
getopts('d:D:Q:G:l:', \%opts);
die(qq/
Usage: samtools.pl pileup2fq [options] <in.cns-pileup>
-D INT maximum depth [$opts{D}]
-Q INT min RMS mapQ [$opts{Q}]
-G INT minimum indel score [$opts{G}]
- -l INT indel filter winsize [$opts{l}]
+ -l INT indel filter winsize [$opts{l}]\n
/) if (@ARGV == 0 && -t STDIN);
my ($last_chr, $seq, $qual, @gaps, $last_pos);
}
}
+#
+# sam2fq
+#
+
+sub sam2fq {
+ my %opts = (n=>20, p=>'');
+ getopts('n:p:', \%opts);
+ die("Usage: samtools.pl sam2fq [-n 20] [-p <prefix>] <inp.sam>\n") if (@ARGV == 0 && -t STDIN);
+ if ($opts{p} && $opts{n} > 1) {
+ my $pre = $opts{p};
+ my @fh;
+ for (0 .. $opts{n}-1) {
+ open($fh[$_], sprintf("| gzip > $pre.%.3d.fq.gz", $_)) || die;
+ }
+ my $i = 0;
+ while (<>) {
+ next if (/^@/);
+ chomp;
+ my @t = split("\t");
+ next if ($t[9] eq '*');
+ my ($name, $seq, $qual);
+ if ($t[1] & 16) { # reverse strand
+ $seq = reverse($t[9]);
+ $qual = reverse($t[10]);
+ $seq =~ tr/ACGTacgt/TGCAtgca/;
+ } else {
+ ($seq, $qual) = @t[9,10];
+ }
+ $name = $t[0];
+ $name .= "/1" if ($t[1] & 0x40);
+ $name .= "/2" if ($t[1] & 0x80);
+ print {$fh[$i]} "\@$name\n$seq\n";
+ if ($qual ne '*') {
+ print {$fh[$i]} "+\n$qual\n";
+ }
+ $i = 0 if (++$i == $opts{n});
+ }
+ close($fh[$_]) for (0 .. $opts{n}-1);
+ } else {
+ die("To be implemented.\n");
+ }
+}
+
+#
+# sra2hdr
+#
+
+# This subroutine does not use an XML parser. It requires that the SRA
+# XML files are properly formated.
+sub sra2hdr {
+ my %opts = ();
+ getopts('', \%opts);
+ die("Usage: samtools.pl sra2hdr <SRA.prefix>\n") if (@ARGV == 0);
+ my $pre = $ARGV[0];
+ my $fh;
+ # read sample
+ my $sample = 'UNKNOWN';
+ open($fh, "$pre.sample.xml") || die;
+ while (<$fh>) {
+ $sample = $1 if (/<SAMPLE.*alias="([^"]+)"/i);
+ }
+ close($fh);
+ # read experiment
+ my (%exp2lib, $exp);
+ open($fh, "$pre.experiment.xml") || die;
+ while (<$fh>) {
+ if (/<EXPERIMENT.*accession="([^\s"]+)"/i) {
+ $exp = $1;
+ } elsif (/<LIBRARY_NAME>\s*(\S+)\s*<\/LIBRARY_NAME>/i) {
+ $exp2lib{$exp} = $1;
+ }
+ }
+ close($fh);
+ # read run
+ my ($run, @fn);
+ open($fh, "$pre.run.xml") || die;
+ while (<$fh>) {
+ if (/<RUN.*accession="([^\s"]+)"/i) {
+ $run = $1; @fn = ();
+ } elsif (/<EXPERIMENT_REF.*accession="([^\s"]+)"/i) {
+ print "\@RG\tID:$run\tSM:$sample\tLB:$exp2lib{$1}\n";
+ } elsif (/<FILE.*filename="([^\s"]+)"/i) {
+ push(@fn, $1);
+ } elsif (/<\/RUN>/i) {
+ if (@fn == 1) {
+ print STDERR "$fn[0]\t$run\n";
+ } else {
+ for (0 .. $#fn) {
+ print STDERR "$fn[$_]\t$run", "_", $_+1, "\n";
+ }
+ }
+ }
+ }
+ close($fh);
+}
+
+#
+# unique
+#
+
+sub unique {
+ my %opts = (f=>250.0, q=>5, r=>2, a=>1, b=>3);
+ getopts('Qf:q:r:a:b:m', \%opts);
+ die("Usage: samtools.pl unique [-f $opts{f}] <in.sam>\n") if (@ARGV == 0 && -t STDIN);
+ my $last = '';
+ my $recal_Q = !defined($opts{Q});
+ my $multi_only = defined($opts{m});
+ my @a;
+ while (<>) {
+ my $score = -1;
+ print $_ if (/^\@/);
+ $score = $1 if (/AS:i:(\d+)/);
+ my @t = split("\t");
+ next if (@t < 11);
+ if ($score < 0) { # AS tag is unavailable
+ my $cigar = $t[5];
+ my ($mm, $go, $ge) = (0, 0, 0);
+ $cigar =~ s/(\d+)[ID]/++$go,$ge+=$1/eg;
+ $cigar = $t[5];
+ $cigar =~ s/(\d+)M/$mm+=$1/eg;
+ $score = $mm * $opts{a} - $go * $opts{q} - $ge * $opts{r}; # no mismatches...
+ }
+ $score = 1 if ($score < 1);
+ if ($t[0] ne $last) {
+ &unique_aux(\@a, $opts{f}, $recal_Q, $multi_only) if (@a);
+ $last = $t[0];
+ }
+ push(@a, [$score, \@t]);
+ }
+ &unique_aux(\@a, $opts{f}, $recal_Q, $multi_only) if (@a);
+}
+
+sub unique_aux {
+ my ($a, $fac, $is_recal, $multi_only) = @_;
+ my ($max, $max2, $max_i) = (0, 0, -1);
+ for (my $i = 0; $i < @$a; ++$i) {
+ if ($a->[$i][0] > $max) {
+ $max2 = $max; $max = $a->[$i][0]; $max_i = $i;
+ } elsif ($a->[$i][0] > $max2) {
+ $max2 = $a->[$i][0];
+ }
+ }
+ if ($is_recal) {
+ if (!$multi_only || @$a > 1) {
+ my $q = int($fac * ($max - $max2) / $max + .499);
+ $q = 250 if ($q > 250);
+ $a->[$max_i][1][4] = $q < 250? $q : 250;
+ }
+ }
+ print join("\t", @{$a->[$max_i][1]});
+ @$a = ();
+}
+
+#
+# uniqcmp: compare two SAM files
+#
+
+sub uniqcmp {
+ my %opts = (q=>10, s=>100);
+ getopts('pq:s:', \%opts);
+ die("Usage: samtools.pl uniqcmp <in1.sam> <in2.sam>\n") if (@ARGV < 2);
+ my ($fh, %a);
+ warn("[uniqcmp] read the first file...\n");
+ &uniqcmp_aux($ARGV[0], \%a, 0);
+ warn("[uniqcmp] read the second file...\n");
+ &uniqcmp_aux($ARGV[1], \%a, 1);
+ warn("[uniqcmp] stats...\n");
+ my @cnt;
+ $cnt[$_] = 0 for (0..9);
+ for my $x (keys %a) {
+ my $p = $a{$x};
+ my $z;
+ if (defined($p->[0]) && defined($p->[1])) {
+ $z = ($p->[0][0] == $p->[1][0] && $p->[0][1] eq $p->[1][1] && abs($p->[0][2] - $p->[1][2]) < $opts{s})? 0 : 1;
+ if ($p->[0][3] >= $opts{q} && $p->[1][3] >= $opts{q}) {
+ ++$cnt[$z*3+0];
+ } elsif ($p->[0][3] >= $opts{q}) {
+ ++$cnt[$z*3+1];
+ } elsif ($p->[1][3] >= $opts{q}) {
+ ++$cnt[$z*3+2];
+ }
+ print STDERR "$x\t$p->[0][1]:$p->[0][2]\t$p->[0][3]\t$p->[0][4]\t$p->[1][1]:$p->[1][2]\t$p->[1][3]\t$p->[1][4]\t",
+ $p->[0][5]-$p->[1][5], "\n" if ($z && defined($opts{p}) && ($p->[0][3] >= $opts{q} || $p->[1][3] >= $opts{q}));
+ } elsif (defined($p->[0])) {
+ ++$cnt[$p->[0][3]>=$opts{q}? 6 : 7];
+ print STDERR "$x\t$p->[0][1]:$p->[0][2]\t$p->[0][3]\t$p->[0][4]\t*\t0\t*\t",
+ $p->[0][5], "\n" if (defined($opts{p}) && $p->[0][3] >= $opts{q});
+ } else {
+ print STDERR "$x\t*\t0\t*\t$p->[1][1]:$p->[1][2]\t$p->[1][3]\t$p->[1][4]\t",
+ -$p->[1][5], "\n" if (defined($opts{p}) && $p->[1][3] >= $opts{q});
+ ++$cnt[$p->[1][3]>=$opts{q}? 8 : 9];
+ }
+ }
+ print "Consistent (high, high): $cnt[0]\n";
+ print "Consistent (high, low ): $cnt[1]\n";
+ print "Consistent (low , high): $cnt[2]\n";
+ print "Inconsistent (high, high): $cnt[3]\n";
+ print "Inconsistent (high, low ): $cnt[4]\n";
+ print "Inconsistent (low , high): $cnt[5]\n";
+ print "Second missing (high): $cnt[6]\n";
+ print "Second missing (low ): $cnt[7]\n";
+ print "First missing (high): $cnt[8]\n";
+ print "First missing (low ): $cnt[9]\n";
+}
+
+sub uniqcmp_aux {
+ my ($fn, $a, $which) = @_;
+ my $fh;
+ $fn = "samtools view $fn |" if ($fn =~ /\.bam/);
+ open($fh, $fn) || die;
+ while (<$fh>) {
+ my @t = split;
+ next if (@t < 11);
+# my $l = ($t[5] =~ /^(\d+)S/)? $1 : 0;
+ my $l = 0;
+ my ($x, $nm) = (0, 0);
+ $nm = $1 if (/NM:i:(\d+)/);
+ $_ = $t[5];
+ s/(\d+)[MI]/$x+=$1/eg;
+ @{$a->{$t[0]}[$which]} = (($t[1]&0x10)? 1 : 0, $t[2], $t[3]-$l, $t[4], "$x:$nm", $x - 4 * $nm);
+ }
+ close($fh);
+}
+
+sub plp2vcf {
+ while (<>) {
+ my @t = split;
+ next if ($t[3] eq '*/*');
+ if ($t[2] eq '*') { # indel
+ my @s = split("/", $t[3]);
+ my (@a, @b);
+ my ($ref, $alt);
+ for (@s) {
+ next if ($_ eq '*');
+ if (/^-/) {
+ push(@a, 'N'.substr($_, 1));
+ push(@b, 'N');
+ } elsif (/^\+/) {
+ push(@a, 'N');
+ push(@b, 'N'.substr($_, 1));
+ }
+ }
+ if ($a[0] && $a[1]) {
+ if (length($a[0]) < length($a[1])) {
+ $ref = $a[1];
+ $alt = ($b[0] . ('N' x (length($a[1]) - length($a[0])))) . ",$b[1]";
+ } elsif (length($a[0]) > length($a[1])) {
+ $ref = $a[0];
+ $alt = ($b[1] . ('N' x (length($a[0]) - length($a[1])))) . ",$b[0]";
+ } else {
+ $ref = $a[0];
+ $alt = ($b[0] eq $b[1])? $b[0] : "$b[0],$b[1]";
+ }
+ } else {
+ $ref = $a[0]; $alt = $b[0];
+ }
+ print join("\t", @t[0,1], '.', $ref, $alt, $t[5], '.', '.'), "\n";
+ } else { # SNP
+ }
+ }
+}
+
#
# Usage
#
Version: $version
Contact: Heng Li <lh3\@sanger.ac.uk>\n
Usage: samtools.pl <command> [<arguments>]\n
-Command: indelFilter filter indels generated by `pileup -c'
- snpFilter filter SNPs generated by `pileup -c'
+Command: varFilter filtering SNPs and short indels
pileup2fq generate fastq from `pileup -c'
showALEN print alignment length (ALEN) following CIGAR
\n/);
projects / samtools.git / blobdiff