var2vcf_paired.pl

#!/usr/bin/env perl

use warnings;
use Getopt::Std;
use strict;

our ($opt_d, $opt_v, $opt_f, $opt_h, $opt_H,
	$opt_p, $opt_q, $opt_F, $opt_S, $opt_Q,
	$opt_o, $opt_C, $opt_M, $opt_P, $opt_N,
	$opt_I, $opt_m, $opt_c, $opt_D, $opt_t,
	$opt_r, $opt_O, $opt_X, $opt_k, $opt_V,
	$opt_x, $opt_A, $opt_b, $opt_G);

our $VERSION = "1.8.2";

getopts('htHSCMAd:v:f:p:q:F:Q:o:P:N:m:c:I:D:r:O:X:k:V:x:b:G:') || Usage();
($opt_h || $opt_H) && Usage();

my $MinDepth = $opt_d ? $opt_d : 5;
my $VarDepth = $opt_v ? $opt_v : 3;
my $FREQ = defined($opt_f) ? $opt_f : 0.02;
my $PMEAN = defined($opt_p) ? $opt_p : 8;
my $QMEAN = defined($opt_q) ? $opt_q : 22.5; # base quality
my $MQMEAN = defined($opt_Q) ? $opt_Q : 0; # mapping quality
my $GTFREQ = defined($opt_F) ? $opt_F : 0.2; # Genotype frequency
my $SN = defined($opt_o) ? $opt_o : 1.5; # Signal to Noise
my $PVAL = defined($opt_P) ? $opt_P : 0.05; # the p-value from fisher test
my $DIFF = defined($opt_D) ? $opt_D : 0.2;
$opt_I = $opt_I ? $opt_I : 12;
$opt_m = $opt_m ? $opt_m : 5.25;
$opt_c = $opt_c ? $opt_c : 0;

my %hash;
my $sample="tumor";
while(<>) {
    chomp;
    next if (/R_HOME/);
    my @a = split(/\t/);
    $sample = $a[0];
    my $chr = $a[2];
    push( @{ $hash{ $chr }->{ $a[3] } }, \@a );
}
my $samplem = "${sample}-match";

if ( $opt_N ) {
    ($sample, $samplem) = split(/\|/, $opt_N);
    $samplem = "${sample}-match" unless( $samplem );
}
(my $sample_nowhitespace = $sample) =~ s/\s/_/g;

print <<VCFHEADER;
##fileformat=VCFv4.2
##source=VarDict_v$VERSION
VCFHEADER

print_reference($opt_G);
print_contigs($opt_b);

print <<VCFHEADER;
##INFO=<ID=SAMPLE,Number=1,Type=String,Description="Sample name (with whitespace translated to underscores)">
##INFO=<ID=TYPE,Number=1,Type=String,Description="Variant Type: SNV Insertion Deletion Complex">
##INFO=<ID=DP,Number=1,Type=Integer,Description="Total Depth">
##INFO=<ID=END,Number=1,Type=Integer,Description="Chr End Position">
##INFO=<ID=VD,Number=1,Type=Integer,Description="Variant Depth">
##INFO=<ID=AF,Number=A,Type=Float,Description="Allele Frequency">
##INFO=<ID=SHIFT3,Number=1,Type=Integer,Description="No. of bases to be shifted to 3 prime for deletions due to alternative alignment">
##INFO=<ID=MSI,Number=1,Type=Float,Description="MicroSatellite. > 1 indicates MSI">
##INFO=<ID=MSILEN,Number=1,Type=Float,Description="MSI unit repeat length in bp">
##INFO=<ID=SSF,Number=1,Type=Float,Description="P-value">
##INFO=<ID=SOR,Number=1,Type=Float,Description="Odds ratio">
##INFO=<ID=LSEQ,Number=1,Type=String,Description="5' flanking seq">
##INFO=<ID=RSEQ,Number=1,Type=String,Description="3' flanking seq">
##INFO=<ID=STATUS,Number=1,Type=String,Description="Somatic or germline status">
##INFO=<ID=P0.01Likely,Number=0,Type=Flag,Description="Likely candidate but p-value > 0.01/5**vd2 (means the evidence in tumor sample might be weak, e.g. small diff in AF)">
##INFO=<ID=InDelLikely,Number=0,Type=Flag,Description="Likely indels more than 2bp are not considered somatic (weak evidence of presence in normal samples)">
##FILTER=<ID=q$QMEAN,Description="Mean Base Quality Below $QMEAN">
##FILTER=<ID=Q$MQMEAN,Description="Mean Mapping Quality Below $MQMEAN">
##FILTER=<ID=p$PMEAN,Description="Mean Position in Reads Less than $PMEAN">
##FILTER=<ID=SN$SN,Description="Signal to Noise Less than $SN">
##FILTER=<ID=Bias,Description="Strand Bias">
##FILTER=<ID=pSTD,Description="Position in Reads has STD of 0">
##FILTER=<ID=MAF0.05,Description="Matched sample has AF > 0.05, thus not somatic">
##FILTER=<ID=d$MinDepth,Description="Total Depth < $MinDepth">
##FILTER=<ID=v$VarDepth,Description="Var Depth < $VarDepth">
##FILTER=<ID=f$FREQ,Description="Allele frequency < $FREQ">
##FILTER=<ID=P$PVAL,Description="Not significant with p-value > $PVAL">
##FILTER=<ID=DIFF$DIFF,Description="Non-somatic or LOH and allele frequency difference < $DIFF">
##FILTER=<ID=MSI$opt_I,Description="Variant in MSI region with $opt_I non-monomer MSI or 12 monomer MSI">
##FILTER=<ID=NM$opt_m,Description="Mean mismatches in reads >= $opt_m, thus likely false positive">
##FILTER=<ID=InGap,Description="The somatic variant is in the deletion gap, thus likely false positive">
##FILTER=<ID=InIns,Description="The somatic variant is adjacent to an insertion variant">
##FILTER=<ID=Cluster${opt_c}bp,Description="Two somatic variants are within $opt_c bp">
##FILTER=<ID=LongAT,Description="The somatic variant is flanked by long A/T (>=14)">
##FORMAT=<ID=GT,Number=1,Type=String,Description="Genotype">
##FORMAT=<ID=DP,Number=1,Type=Integer,Description="Total Depth">
##FORMAT=<ID=VD,Number=1,Type=Integer,Description="Variant Depth">
##FORMAT=<ID=AD,Number=R,Type=Integer,Description="Allelic depths for the ref and alt alleles in the order listed">
##FORMAT=<ID=ALD,Number=2,Type=Integer,Description="Variant forward, reverse reads">
##FORMAT=<ID=RD,Number=2,Type=Integer,Description="Reference forward, reverse reads">
##FORMAT=<ID=AF,Number=A,Type=Float,Description="Allele Frequency">
##FORMAT=<ID=ADJAF,Number=1,Type=Float,Description="Adjusted AF for indels due to local realignment">
##FORMAT=<ID=BIAS,Number=2,Type=String,Description="Strand Bias Info">
##FORMAT=<ID=PMEAN,Number=1,Type=Float,Description="The mean distance to the nearest 5 or 3 prime read end (whichever is closer) in all reads that support the variant call">
##FORMAT=<ID=PSTD,Number=1,Type=Float,Description="Position STD in reads">
##FORMAT=<ID=QUAL,Number=1,Type=Float,Description="Mean quality score in reads">
##FORMAT=<ID=QSTD,Number=1,Type=Float,Description="Quality score STD in reads">
##FORMAT=<ID=SBF,Number=1,Type=Float,Description="Strand Bias Fisher p-value">
##FORMAT=<ID=ODDRATIO,Number=1,Type=Float,Description="Strand Bias Odds ratio">
##FORMAT=<ID=MQ,Number=1,Type=Integer,Description="Mean Mapping Quality">
##FORMAT=<ID=SN,Number=1,Type=Float,Description="Signal to noise">
##FORMAT=<ID=HIAF,Number=1,Type=Float,Description="Allele frequency using only high quality bases">
##FORMAT=<ID=NM,Number=1,Type=Float,Description="Mean mismatches in reads">
VCFHEADER

print join("\t", "#CHROM", qw(POS ID REF ALT QUAL FILTER INFO FORMAT), $sample, $samplem), "\n";

# Exit if we don't have any variants to write 
exit(0) unless( %hash );

my @chrs = reorder(keys %hash);
foreach my $chr (@chrs) {
    my @pos = sort { $a <=> $b } (keys %{ $hash{ $chr } });
    my ($pds, $pde) = (0, 0); # previous Deletion variant's start and end
    my ($pis, $pie) = (0, 0); # previous Insertion variant's start and end
    my ($pvs, $pve) = (0, 0); # previous SNV variant's start and end
    my ($pinfo1, $pfilter, $pinfo2) = ("", "", "");
    foreach my $p (@pos) {
	my @tmp = sort { $b->[14] <=> $a->[14] } @{ $hash{ $chr }->{ $p } };
	my $ALL = $opt_A ? @tmp + 0 : 1;
	my %seen = ();
	for(my $i = 0; $i < $ALL; $i++) {
	    my $d = $tmp[$i]; # Only the highest AF get represented
	    my ($sample, $gene, $chrt, $start, $end, $ref, $alt, $dp1, $vd1, $rfwd1, $rrev1, $vfwd1, $vrev1, $gt1, $af1, $bias1, $pmean1, $pstd1, $qual1, $qstd1, $mapq1, $sn1, $hiaf1, $adjaf1, $nm1, $sbf1, $oddratio1, $dp2, $vd2, $rfwd2, $rrev2, $vfwd2, $vrev2, $gt2, $af2, $bias2, $pmean2, $pstd2, $qual2, $qstd2, $mapq2, $sn2, $hiaf2, $adjaf2, $nm2, $sbf2, $oddratio2, $shift3, $msi, $msilen, $lseq, $rseq, $seg, $status, $type, $sv1, $duprate1, $sv2, $duprate2, $pvalue, $oddratio)  = @$d;
	    my $rd1 = $rfwd1 + $rrev1;
	    my $rd2 = $rfwd2 + $rrev2;
        next unless ( $ref );
	    next if ( $seen{ "$chrt-$start-$end-$ref-$alt" } );
	    $seen{ "$chrt-$start-$end-$ref-$alt" } = 1;
	    unless ($type) { $type = "REF"; }
	    #$pvalue *= sqrt(60/($mapq1+length($ref)+length($alt)-1))*$af1;
        my @filters = ();
        my @filters2 = ();
        if ( $oddratio eq "Inf" ) {
            $oddratio = 0;
        }
	    if ( $oddratio1 eq "Inf" ) {
		$oddratio1 = 0;
	    } elsif ( $oddratio1 < 1 && $oddratio1 > 0 ) {
		$oddratio1 = sprintf("%.2f", 1/$oddratio1);
	    }
	    if ( $oddratio2 eq "Inf" ) {
		$oddratio2 = 0;
	    } elsif ( $oddratio2 < 1 && $oddratio2 > 0 ) {
		$oddratio2 = sprintf("%.2f", 1/$oddratio2);
	    }
	    if ($dp1 < $MinDepth) {
		push( @filters, "d$MinDepth") unless ( $status eq "StrongSomatic" && $pvalue < 0.15 && $af1*$vd1 >= 0.5);
	    }
	    if ($vd1 < $VarDepth) {
		push( @filters, "v$VarDepth") unless ( $status eq "StrongSomatic" && $pvalue < 0.15 && $af1*$vd1 >= 0.5);
	    }
	    push(@filters2, "d$MinDepth") if ( $dp2 < $MinDepth );
	    push(@filters2, "v$VarDepth") if ( $vd2 < $VarDepth );
	    #if ( $status =~ /Somatic/ || $status =~ /SampleSpecific/ ) {
		push( @filters, "f$FREQ") if ($af1 < $FREQ);
		#push( @filters, "MAF0.05") if ($qual2 >= $QMEAN && $pmean2 >= $PMEAN && $mapq2 >= $MQMEAN && $sn2 >= $SN && $nm2 < $opt_m && $af2 > 0.05);
		push( @filters, "p$PMEAN") if ($pmean1 < $PMEAN);
		push( @filters, "pSTD") if ($pstd1 == 0 && $vd1 < $MinDepth);
		push( @filters, "q$QMEAN") if ($qual1 < $QMEAN);
		push( @filters, "Q$MQMEAN") if ($mapq1 < $MQMEAN);
		push( @filters, "Q$MQMEAN") if ($mapq1 < 10 && $type eq "SNV"); # consider SNV somatic in low mapping quality region false positves
		push( @filters, "SN$SN") if ($sn1 < $SN);
		push( @filters, "NM$opt_m") if ($nm1 >= $opt_m);
		#push( @filters, "Bias") if (($bias1 eq "2;1" || $bias1 eq "2;0") && $sbf1 < 0.01 && ($oddratio1 > 5 || $oddratio1 == 0));
		push( @filters, "Bias") if ($bias1 eq "2;1" && $sbf1 < 0.01 && ($oddratio1 > 5 || $oddratio1 == 0) && $end - $start < 100);
	    #} elsif ( $status =~ /LOH/ || $status =~ /Deletion/ ) {
		push( @filters2, "f$FREQ") if ($af2 < $FREQ);
		push( @filters2, "p$PMEAN") if ($pmean2 < $PMEAN);
		push( @filters2, "pSTD") if ($pstd2 == 0 && $vd2 < $MinDepth);
		push( @filters2, "q$QMEAN") if ($qual2 < $QMEAN);
		push( @filters2, "Q$MQMEAN") if ($mapq2 < $MQMEAN);
		push( @filters2, "SN$SN") if ($sn2 < $SN);
		push( @filters2, "NM$opt_m") if ($nm2 >= $opt_m);
		#push( @filters2, "Bias") if (($bias2 eq "2;1" || $bias2 eq "2;0") && $sbf2 < 0.01 && ($oddratio2 > 5 || $oddratio2 == 0));
		my %bias_filters = map { $_, 1 } @filters;
		push( @filters, "Bias") if (!$bias_filters{ "Bias" } && $bias2 eq "2;1" && $sbf2 < 0.01 && ($oddratio2 > 5 || $oddratio2 == 0) && $end - $start < 100);
	    #}
	    # Require stringent statistics in regions with MSI
	    if ( ($msi > $opt_I && $msilen > 1) || ($msi > 12 && $msilen == 1)) {
		    push( @filters, "MSI$opt_I") unless( $status eq "StrongSomatic" && $pvalue < 0.0005 );
	    }
	    if ( abs(length($ref)-length($alt)) == $msilen && !grep(/^MSI$opt_I/,@filters)) {
		push( @filters, "MSI$opt_I") if ( ($msi > $opt_I && $msilen > 1 && $af1 < 0.35 && $af2 < 0.35) || ($msi > 12 && $msilen == 1 && $af1 < 0.35 && $af2 < 0.35) );
	    }
	    my $p_likely = 0;
	    my $indel_likely = 0;
	    #push( @filters, "Bias") if (($a[15] eq "2;1" && $a[24] < 0.01) || ($a[15] eq "2;0" && $a[24] < 0.01) ); #|| ($a[9]+$a[10] > 0 && abs($a[9]/($a[9]+$a[10])-$a[11]/($a[11]+$a[12])) > 0.5));
	    if ( $opt_M ) {
		if ( $pvalue > $PVAL ) {
		    push(@filters, "P$PVAL") unless ($status eq "StrongSomatic" && (($pvalue < 0.25 && $af1 > 0.1 ) || ($pvalue < 0.5 && $af1 > 0.20) || ($pvalue < 0.15 && $af1 > 0.05)));
		} elsif ( $status =~ /LikelySomatic/ && $pvalue > 0.05/5**$vd2 ) { # Increase the stringency for LikelySomatic
		    $p_likely = 1;
		} elsif ( $status =~ /Likely/ && $type ne "SNV" ) {
		    $indel_likely = 1 unless(length($ref) <= 2 && length($alt) <= 2);
		}
	    }
	    #if ( @filters == 0 && abs(length($ref)-length($alt)) == $msilen ) {
		#push( @filters, "InGap" ) if ( $pds && $type eq "SNV" && $start <= $pde && $end >= $pds && $status =~ /Somatic/ );
		#push( @filters, "InIns" ) if ( $pis && $type eq "SNV" && $start <= $pie && $end >= $pis && $status =~ /Somatic/ );
		#push( @filters, "LongAT") if (isLongAT($lseq) || isLongAT($rseq));
	    #}
	    #my $filter = @filters > 1 ? join(";", @filters) : (((@filters == 1 && ($filters[0] eq "P$PVAL" || $filters[0] eq "P0.01Likely" || $filters[0] eq "InDelLikely" || "DIFF$DIFF")) ? "PASS" : $filters[0]) :"PASS");
	    my $filter = @filters > 0 ? join(";", @filters) : "PASS";

	    # Unless somatic only option (-M) is specified, any good variants in germline should be
	    # reported as well, regardless of the tumor sample
	    unless($opt_M) {
		$filter = "PASS" if ( $filter ne "PASS" && @filters2 == 0 );
	    }
	    my $gt = (1-$af1 < $GTFREQ) ? "1/1" : ($af1 >= 0.5 ? "1/0" : ($af1 >= $FREQ ? "0/1" : "0/0"));
	    my $gtm = (1-$af2 < $GTFREQ) ? "1/1" : ($af2 >= 0.5 ? "1/0" : ($af2 >= $FREQ ? "0/1" : "0/0"));
        $bias1 =~ s/;/,/;
        $bias2 =~ s/;/,/;
        $bias1 = "0,0" if ($bias1 eq '0');
        $bias2 = "0,0" if ($bias2 eq '0');
        $mapq1 = sprintf '%.0f', $mapq1;
        $mapq2 = sprintf '%.0f', $mapq2;
	    my $qual = $vd1 > $vd2 ? int(log($vd1)/log(2) * $qual1) : int(log($vd2)/log(2) * $qual2);
	    if ( $pfilter eq "PASS" && $pinfo2 =~ /Somatic/ && $pinfo2 =~ /TYPE=SNV/ && $filter eq "PASS" && $status =~ /Somatic/ && $type eq "SNV" && $start - $pvs < $opt_c ) {
		$pfilter = "Cluster${opt_c}bp";
		$filter = "Cluster${opt_c}bp";
	    }
	    if ( $pinfo1 ) {
		#print "$pinfo1\t$pfilter\t$pinfo2\n" unless ( ($opt_M && $pinfo2 !~ /Somatic/) || $opt_S && $pfilter ne "PASS" );
		print "$pinfo1\t$pfilter\t$pinfo2\n" unless ( $opt_S && $pfilter ne "PASS" );
	    }
	    ($pinfo1, $pfilter, $pinfo2) = (join("\t", $chr, $start, ".", $ref, $alt, $qual), $filter,
		join("\t", join("","STATUS=$status;SAMPLE=$sample_nowhitespace;TYPE=$type;DP=$dp1;VD=$vd1;AF=$af1;SHIFT3=$shift3;MSI=$msi;MSILEN=$msilen;SSF=$pvalue;SOR=$oddratio;LSEQ=$lseq;RSEQ=$rseq",
		     $p_likely ? ";P0.01Likely" : "", $indel_likely ?  ";InDelLikely" : ""),
		    "GT:DP:VD:ALD:RD:AD:AF:BIAS:PMEAN:PSTD:QUAL:QSTD:SBF:ODDRATIO:MQ:SN:HIAF:ADJAF:NM",
		    "$gt:$dp1:$vd1:$vfwd1,$vrev1:$rfwd1,$rrev1:$rd1,$vd1:$af1:$bias1:$pmean1:$pstd1:$qual1:$qstd1:$sbf1:$oddratio1:$mapq1:$sn1:$hiaf1:$adjaf1:$nm1",
		    "$gtm:$dp2:$vd2:$vfwd2,$vrev2:$rfwd2,$rrev2:$rd2,$vd2:$af2:$bias2:$pmean2:$pstd2:$qual2:$qstd2:$sbf2:$oddratio2:$mapq2:$sn2:$hiaf2:$adjaf2:$nm2"));
	    ($pds, $pde) = ($start+1, $end) if ($type eq "Deletion");
	    ($pis, $pie) = ($start-1, $end+1) if ($type eq "Insertion");
	    ($pvs, $pve) = ($start, $end) if ( $type eq "SNV" && $filter eq "PASS");
	}
    }
    if ( $pinfo1 ) {
	print "$pinfo1\t$pfilter\t$pinfo2\n" unless ( $opt_S && $pfilter ne "PASS" );
    }
}

sub isLongAT {
    my $seq = shift;
    return 1 if ( $seq =~ /T{14,}/ );
    return 1 if ( $seq =~ /A{14,}/ );
    return 0;
}

sub reorder {
    my @chr = @_;
    my @chrn = (); # numeric chromosomes
    my @nonchrn = (); # non-numeric chrosomes
    foreach my $c (@chr) {
        if ( $c =~ /\d/ && $c !~ /_/) {
            my $t = $c;
            $t =~ s/\D//g;
            push(@chrn, [$t, $c]);
        } else {
        	next if ( $c eq "X" || $c eq "chrX" ||  $c eq "Y" ||  $c eq "chrY" );
            next if ( $c eq "MT" || $c eq "chrM" );
            push(@nonchrn, $c);
        }
    }
    @chrn = sort { $a->[0] <=> $b->[0]; } @chrn;
    @chr = map { $_->[1]; } @chrn;
    if ( $hash{ X } ) {
        push(@chr, 'X' );
    } elsif ( $hash{ chrX } ) {
        push(@chr, 'chrX' );
    } 
    if ( $hash{ Y } ) {
        push(@chr, 'Y' );
    } elsif ( $hash{ chrY } ) {
        push(@chr, 'chrY' );
    } 
    if ( $hash{ MT } ) {
        push(@chr, 'MT' );
    } elsif ( $hash{ chrM } ) {
        push(@chr, 'chrM' );
    }
    push ( @chr, @nonchrn );
    return (@chr);
}

sub print_contigs
{
	my ($path) = @_;
	if (not defined($path)) {return;}

	open(my $bed_file, "<", $path)
		or return;

	while (my $line = <$bed_file>)
	{
		chomp $line;
		my ($name, $start, $end) = split(/\t/, $line);
		print "##contig=<ID=${name},length=${end}>\n";
	}
}

sub print_reference {
	my $path = shift;
	return unless defined($path);
	print "##reference=$path\n";
}

sub Usage {
print <<USAGE;
$0 [-hHS] [-p pos] [-q qual] [-d depth] [-v depth] [-f frequency] [-F frequency] vars.txt
Version: $VERSION
The program will convert the variant output from checkVar.pl script into validated VCF file.

Options are:

    -h	Print this usage.
    -H	Print this usage.
    -C  If set, chrosomes will have names of 1,2,3,...,X,Y, instead of chr1, chr2, ..., chrX, chrY
    -S	If set, variants that didn't pass filters will not be present in VCF file
    -M  If set, will increase stringency for candidate somatic: flag P0.01Likely and InDelLikely, and add filter P0.05
    -A  Indicate to output all variants at the same position.  By default, only the variant with the highest allele frequency is converted to VCF.
    -D  float (0-1) # Deprecated
        The minimum allele frequency difference between two samples required in addition to p-value.  Not compatible
	with -M option.  It's for interest of identifying variants with different AF, not just somatic.
    -c  int
        If two somatic candidates are within {int} bp, they're both filtered.  Default: 0 or no filtering
    -I  int
        The maximum non-monomer MSI allowed for a HT variant with AF < 0.6.  By default, 12, or any variants with AF < 0.6 in a region
	with > 12 non-monomer MSI will be considered false positive.  For monomers, that number is 10.
    -m  int
        The maximum mean mismatches allowed.  Default: 5.25, or if a variant is supported by reads with more than 5.25 mismatches, it'll be considered
	false positive.  Mismatches don't includes indels in the alignment.
    -N  Name(s)
        The sample name(s).  If only one name is given, the matched will be simply names as "name-match".  Two names
	are given separated by "|", such as "tumor|blood".
    -P  float
        The maximum p-value.  Default to 0.05.
    -p	float
    	The minimum mean position of variants in the read.  Default: 5.
    -q	float
    	The minimum mean base quality.  Default to 22.5 for Illumina sequencing
    -Q	float
    	The minimum mapping quality.  Default to 0 for Illumina sequencing
    -d	integer
    	The minimum total depth.  Default to 5
    -v	integer
    	The minimum variant depth.  Default to 3
    -f	float
    	The minimum allele frequency.  Default to 0.02
    -o	signal/noise
    	The minimum signal to noise, or the ratio of hi/(lo+0.5).  Default to 1.5.  Set it higher for deep sequencing.
    -F	float
    	The minimum allele frequency to consider to be homozygous.  Default to 0.2.  Thus frequency > 0.8 (1-0.2) will 
	be considered homozygous "1/1", between 0.5 - (1-0.2) will be "1/0", between (-f) - 0.5 will be "0/1",
	below (-f) will be "0/0".
	-b  Path to the *.bed file which is used to generate contigs in the header
	-G  Path to the *.fasta (*.fa) file which is used to generate reference tag in the header

AUTHOR
       Written by Zhongwu Lai, AstraZeneca, Boston, USA

REPORTING BUGS
       Report bugs to zhongwu\@yahoo.com

COPYRIGHT
       This is free software: you are free to change and redistribute it.  There is NO WARRANTY, to the extent permitted by law.

USAGE
exit(0);
}