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fpfilter.pl
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fpfilter.pl
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#!/usr/bin/env perl
use warnings;
use strict;
use Getopt::Long;
use FileHandle;
=head1 VERSION
Version 1.01
=head1 SYNOPSIS
fpfilter is an accessory script to VarScan 2 (http://varscan.sourceforge.net) that filters false positives using information from bam-readcounts (https://github.com/genome/bam-readcount)
=head1 USAGE fpfilter.pl [varScan file] [bam-readcounts output] OPTIONS
=head3 COMMANDS:
=cut
# OPTIONS:
# --output-basename The basename for output files. Two will be created: basename.pass and basename.fail
our $VERSION = '1.01';
my $usage = qq{USAGE: fpfilter.pl [varScan file] [bam-readcounts output] OPTIONS
OPTIONS:
--output-basename The basename for output files. Two will be created: basename.pass and basename.fail
};
die $usage if(!$ARGV[1]);
## Define filtering parameters ##
my $min_read_pos = 0.10;
my $max_read_pos = 1 - $min_read_pos;
my $min_var_freq = 0.05;
my $min_var_count = 4;
my $min_strandedness = 0.01;
my $max_strandedness = 1 - $min_strandedness;
my $max_mm_qualsum_diff = 50;
my $max_mapqual_diff = 30;
my $max_readlen_diff = 25;
my $min_var_dist_3 = 0.20;
my $max_var_mm_qualsum = 100;
## Parse arguments ##
my $output_basename = "outfile";
my $verbose = 0;
my $ret = parse_arguments();
execute();
################################################################################
=head2 parse_arguments
=cut
################################################################################
sub parse_arguments
{
# my $output_alignments, my $output_snps, my $output_indels, my $fasta_file;#, my $quality_file;
# my $min_identity, my $default_qual_score, my $min_qual_score, my $primer_trim, my $verbose;
my $result = GetOptions (
"output-basename=s" => \$output_basename,
"verbose=s" => \$verbose,
);
}
################################################################################
=head2 execute [ALIGNMENTS] [OPTIONS]
Run the filter
=cut
sub execute
{
my %stats = ();
$stats{'num_variants'} = $stats{'num_fail_pos'} = $stats{'num_fail_strand'} = $stats{'num_fail_varcount'} = $stats{'num_fail_varfreq'} = $stats{'num_fail_mmqs'} = $stats{'num_fail_var_mmqs'} = $stats{'num_fail_mapqual'} = $stats{'num_fail_readlen'} = $stats{'num_fail_dist3'} = $stats{'num_pass_filter'} = $stats{"num_no_readcounts"} = 0;
## Load the read counts ##
my %readcounts_by_position = ();
my $rc_input = new FileHandle ($ARGV[1]);
my $lineCounter = 0;
while (<$rc_input>)
{
chomp;
my $line = $_;
$lineCounter++;
my ($chrom, $position) = split(/\t/, $line);
$readcounts_by_position{"$chrom\t$position"} = $line;
}
close($rc_input);
## Open the output files ##
# open(PASS, ">$output_basename.pass") or die "Can't open output file: $!\n";
# open(FAIL, ">$output_basename.fail") or die "Can't open output file: $!\n";
## Parse the input file ##
my $input = new FileHandle ($ARGV[0]);
$lineCounter = 0;
while (<$input>)
{
if (/^#/){
print $_;
next;
}
# chomp;
my $line = $_;
$lineCounter++;
my ($chrom, $position, $id, $ref, $var, $qual, $filter, $rest_of_line) = split(/\t/, $line, 8);
$ref = uc($ref);
$var = uc($var);
if(!($var =~ /[ACGT]/)) {
$var = iupac_to_base($ref, $var);
}
$stats{'num_variants'}++;
if($readcounts_by_position{"$chrom\t$position"})
{
my $readcounts = $readcounts_by_position{"$chrom\t$position"};
my $ref_result = read_counts_by_allele($readcounts, $ref);
my $var_result = read_counts_by_allele($readcounts, $var);
if($ref_result && $var_result)
{
## Parse out the bam-readcounts details for each allele. The fields should be: ##
#num_reads : avg_mapqual : avg_basequal : avg_semq : reads_plus : reads_minus : avg_clip_read_pos : avg_mmqs : reads_q2 : avg_dist_to_q2 : avgRLclipped : avg_eff_3'_dist
my ($ref_count, $ref_map_qual, $ref_base_qual, $ref_semq, $ref_plus, $ref_minus, $ref_pos, $ref_subs, $ref_mmqs, $ref_q2_reads, $ref_q2_dist, $ref_avg_rl, $ref_dist_3) = split(/\t/, $ref_result);
my ($var_count, $var_map_qual, $var_base_qual, $var_semq, $var_plus, $var_minus, $var_pos, $var_subs, $var_mmqs, $var_q2_reads, $var_q2_dist, $var_avg_rl, $var_dist_3) = split(/\t/, $var_result);
my $ref_strandedness = my $var_strandedness = 0.50;
$ref_dist_3 = 0.5 if(!$ref_dist_3);
## Use conservative defaults if we can't get mismatch quality sums ##
$ref_mmqs = 50 if(!$ref_mmqs);
$var_mmqs = 0 if(!$var_mmqs);
my $mismatch_qualsum_diff = $var_mmqs - $ref_mmqs;
## Determine map qual diff ##
my $mapqual_diff = $ref_map_qual - $var_map_qual;
## Determine difference in average supporting read length ##
my $readlen_diff = $ref_avg_rl - $var_avg_rl;
## Determine ref strandedness ##
if(($ref_plus + $ref_minus) > 0) {
$ref_strandedness = $ref_plus / ($ref_plus + $ref_minus);
$ref_strandedness = sprintf("%.2f", $ref_strandedness);
}
## Determine var strandedness ##
if(($var_plus + $var_minus) > 0) {
$var_strandedness = $var_plus / ($var_plus + $var_minus);
$var_strandedness = sprintf("%.2f", $var_strandedness);
}
if($var_count && ($var_plus + $var_minus))
{
## We must obtain variant read counts to proceed ##
my $var_freq = $var_count / ($ref_count + $var_count);
## FAILURE 1: READ POSITION ##
if(($var_pos < $min_read_pos)) { # || $var_pos > $max_read_pos)) {
$filter = "ReadPos";
# print FAIL "$line\t$ref_pos\t$var_pos\t$ref_strandedness\t$var_strandedness\tReadPos<$min_read_pos\n";
# print "$line\t$ref_pos\t$var_pos\t$ref_strandedness\t$var_strandedness\tReadPos<$min_read_pos\n"if ($verbose);
$stats{'num_fail_pos'}++;
}
## FAILURE 2: Variant is strand-specific but reference is NOT strand-specific ##
elsif(($var_strandedness < $min_strandedness || $var_strandedness > $max_strandedness) && ($ref_strandedness >= $min_strandedness && $ref_strandedness <= $max_strandedness)) {
## Print failure to output file if desired ##
$filter = "Strandedness";
# print FAIL "$line\t$ref_pos\t$var_pos\t$ref_strandedness\t$var_strandedness\tStrandedness: Ref=$ref_strandedness Var=$var_strandedness\n";
# print "$line\t$ref_pos\t$var_pos\t$ref_strandedness\t$var_strandedness\tStrandedness: Ref=$ref_strandedness Var=$var_strandedness\n"if ($verbose);
$stats{'num_fail_strand'}++;
}
## FAILURE : Variant allele count does not meet minimum ##
elsif($var_count < $min_var_count) {
$filter = "VarCount";
# print FAIL "$line\t$ref_pos\t$var_pos\t$ref_strandedness\t$var_strandedness\tVarCount:$var_count\n";
print "$line\t$ref_pos\t$var_pos\t$ref_strandedness\t$var_strandedness\tVarCount:$var_count\n" if ($verbose);
$stats{'num_fail_varcount'}++;
}
## FAILURE : Variant allele frequency does not meet minimum ##
elsif($var_freq < $min_var_freq) {
$filter = "VarFreq";
# print FAIL "$line\t$ref_pos\t$var_pos\t$ref_strandedness\t$var_strandedness\tVarFreq:$var_freq\n";
# print "$line\t$ref_pos\t$var_pos\t$ref_strandedness\t$var_strandedness\tVarFreq:$var_freq\n" if ($verbose);
$stats{'num_fail_varfreq'}++;
}
## FAILURE 3: Paralog filter for sites where variant allele mismatch-quality-sum is significantly higher than reference allele mmqs
elsif($mismatch_qualsum_diff> $max_mm_qualsum_diff) {
## Print failure to output file if desired ##
$filter = "MismatchQualsum";
# print FAIL "$line\t$ref_pos\t$var_pos\t$ref_strandedness\t$var_strandedness\tMismatchQualsum:$var_mmqs-$ref_mmqs=$mismatch_qualsum_diff\n";
# print "$line\t$ref_pos\t$var_pos\t$ref_strandedness\t$var_strandedness\tMismatchQualsum:$var_mmqs-$ref_mmqs=$mismatch_qualsum_diff" if ($verbose);
$stats{'num_fail_mmqs'}++;
}
## FAILURE 4: Mapping quality difference exceeds allowable maximum ##
elsif($mapqual_diff > $max_mapqual_diff) {
$filter = "MapQual";
# print FAIL "$line\t$ref_pos\t$var_pos\t$ref_strandedness\t$var_strandedness\tMapQual:$ref_map_qual-$var_map_qual=$mapqual_diff\n";
# print "$line\t$ref_pos\t$var_pos\t$ref_strandedness\t$var_strandedness\tMapQual:$ref_map_qual-$var_map_qual=$mapqual_diff" if ($verbose);
$stats{'num_fail_mapqual'}++;
}
## FAILURE 5: Read length difference exceeds allowable maximum ##
elsif($readlen_diff > $max_readlen_diff) {
$filter = "ReadLen";
# print FAIL "$line\t$ref_pos\t$var_pos\t$ref_strandedness\t$var_strandedness\tReadLen:$ref_avg_rl-$var_avg_rl=$readlen_diff\n";
# print "$line\t$ref_pos\t$var_pos\t$ref_strandedness\t$var_strandedness\tReadLen:$ref_avg_rl-$var_avg_rl=$readlen_diff" if ($verbose);
$stats{'num_fail_readlen'}++;
}
## FAILURE 5: Read length difference exceeds allowable maximum ##
elsif($var_dist_3 < $min_var_dist_3) {
$filter = "VarDist3";
# print FAIL "$line\t$ref_pos\t$var_pos\t$ref_strandedness\t$var_strandedness\tVarDist3:$var_dist_3\n";
# print "$line\t$ref_pos\t$var_pos\t$ref_strandedness\t$var_strandedness\tVarDist3:$var_dist_3\n" if ($verbose);
$stats{'num_fail_dist3'}++;
}
elsif($max_var_mm_qualsum && $var_mmqs > $max_var_mm_qualsum) {
$filter = "VarMMQS";
# print FAIL "$line\t$ref_pos\t$var_pos\t$ref_strandedness\t$var_strandedness\tVarMMQS: $var_mmqs > $max_var_mm_qualsum\n";
# print "$line\t$ref_pos\t$var_pos\t$ref_strandedness\t$var_strandedness\tVarMMQS: $var_mmqs > $max_var_mm_qualsum\n" if ($verbose);
$stats{'num_fail_var_mmqs'}++;
}
## SUCCESS: Pass Filter ##
else {
$stats{'num_pass_filter'}++;
## Print output, and append strandedness information ##
# print PASS "$line\n";
# print "$line\tPASS\n" if($verbose);
}
}
}
else
{
$stats{'num_no_readcounts'}++;
$filter = "NoReadCounts";
print STDERR "$line\tno_readcounts\n";
}
}
else
{
$stats{'num_no_readcounts'}++;
$filter = "NoReadCounts";
print STDERR "$line\tno_readcounts\n";
}
print join "\t", ($chrom, $position, $id, $ref, $var, $qual, $filter, $rest_of_line);
}
close($input);
# close(PASS);
# close(FAIL);
## Print filtering stats ##
print STDERR $stats{'num_variants'} . " variants\n";
print STDERR $stats{'num_no_readcounts'} . " failed to get readcounts for variant allele\n";
print STDERR $stats{'num_fail_pos'} . " had read position < $min_read_pos\n";
print STDERR $stats{'num_fail_strand'} . " had strandedness < $min_strandedness\n";
print STDERR $stats{'num_fail_varcount'} . " had var_count < $min_var_count\n";
print STDERR $stats{'num_fail_varfreq'} . " had var_freq < $min_var_freq\n";
print STDERR $stats{'num_fail_mmqs'} . " had mismatch qualsum difference > $max_mm_qualsum_diff\n";
print STDERR $stats{'num_fail_var_mmqs'} . " had variant MMQS > $max_var_mm_qualsum\n" if($stats{'num_fail_var_mmqs'});
print STDERR $stats{'num_fail_mapqual'} . " had mapping quality difference > $max_mapqual_diff\n";
print STDERR $stats{'num_fail_readlen'} . " had read length difference > $max_readlen_diff\n";
print STDERR $stats{'num_fail_dist3'} . " had var_distance_to_3' < $min_var_dist_3\n";
print STDERR $stats{'num_pass_filter'} . " passed the strand filter\n";
return(0);
}
################################################################################
=head3 iupac_to_base
Convert IUPAC ambiguity codes to variant bases
=cut
sub iupac_to_base {
(my $allele1, my $allele2) = @_;
return($allele2) if($allele2 eq "A" || $allele2 eq "C" || $allele2 eq "G" || $allele2 eq "T");
if($allele2 eq "M") {
return("C") if($allele1 eq "A");
return("A") if($allele1 eq "C");
return("A"); ## Default for triallelic variant
} elsif($allele2 eq "R") {
return("G") if($allele1 eq "A");
return("A") if($allele1 eq "G");
return("A"); ## Default for triallelic variant
} elsif($allele2 eq "W") {
return("T") if($allele1 eq "A");
return("A") if($allele1 eq "T");
return("A"); ## Default for triallelic variant
} elsif($allele2 eq "S") {
return("C") if($allele1 eq "G");
return("G") if($allele1 eq "C");
return("C"); ## Default for triallelic variant
} elsif($allele2 eq "Y") {
return("C") if($allele1 eq "T");
return("T") if($allele1 eq "C");
return("C"); ## Default for triallelic variant
} elsif($allele2 eq "K") {
return("G") if($allele1 eq "T");
return("T") if($allele1 eq "G");
return("G"); ## Default for triallelic variant
}
return($allele2);
}
################################################################################
=head3 read_counts_by_allele
Retrieve relevant read counts for a certain allele
=cut
sub read_counts_by_allele {
(my $line, my $allele) = @_;
my @lineContents = split(/\t/, $line);
my $numContents = @lineContents;
for(my $colCounter = 5; $colCounter < $numContents; $colCounter++) {
my $this_allele = $lineContents[$colCounter];
my @alleleContents = split(/\:/, $this_allele);
if($alleleContents[0] eq $allele) {
my $numAlleleContents = @alleleContents;
return("") if($numAlleleContents < 8);
my $return_string = "";
my $return_sum = 0;
for(my $printCounter = 1; $printCounter < $numAlleleContents; $printCounter++) {
$return_sum += $alleleContents[$printCounter];
$return_string .= "\t" if($return_string);
$return_string .= $alleleContents[$printCounter];
}
return($return_string);
}
}
return("");
}
=head1 AUTHOR
Daniel C. Koboldt, << <dkoboldt at genome.wustl.edu> >>
The Genome Institute at Washington University School of Medicine
St. Louis, Missouri, USA
=head1 COPYRIGHT
Copyright 2009-2012 Daniel C. Koboldt and Washington University
All rights reserved.
=head1 LICENSE
This program is free for non-commercial use.
=cut