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BS-Snper.pl
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BS-Snper.pl
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#!/usr/bin/perl -w
=head1 Name
BS-Snper.pl -- Detect the SNP according to the candidate positions
=head1 Version
Author: Shengjie Gao, gaoshengjie@genomics.cn; Dan Zou, zoudan.nudt@gmail.com; Xuesong HU, huxs001@gmail.com.
Version: 1.1, Date: 2018-07-27
=head1 Usage
--variants-only output variant sites only
--regions-file <file> restrict to regions listed in a file
--help output help information to screen
=head1 Exmple
perl BS-Snper.pl --fa hg19.fa --input BSMAP.sort.bam --output snp.candidate.out --methcg meth.cg --methchg meth.chg --methchh meth.chh --minhetfreq 0.1 --minhomfreq 0.85 --minquali 15 --mincover 10 --maxcover 1000 --minread2 2 --errorate 0.02 --mapvalue 20 >SNP.out 2>SNP.log
#perl BS-Snper.pl --fa hg19.fa --input sort.bam --output result --methoutput meth.out --minhetfreq 0.1 --minhomfreq 0.85 --minquali 15 --mincover 10 --maxcover 1000 --minread2 2 --errorate 0.02 >SNP.out 2>SNP.log\n
=cut
use strict;
use Getopt::Long;
use POSIX;
use FindBin '$Bin';
use File::Path; ## function " mkpath" and "rmtree" deal with directory
use File::Basename qw(basename dirname);
my ($Help,$fasta,$bam,$mapvalue,$minhetfreq,$minhomfreq,$minquali,$minread2,$mincover,$maxcover,$errorate,$pvalue,$interval,$output,$methcg,$methchg,$methchh);
my ($regFile,$varOnly);
die `pod2text $0` if @ARGV==0;
GetOptions(
"fa:s"=>\$fasta,
"input:s"=>\$bam,
"regions-file:s"=>\$regFile,
"variants-only"=>\$varOnly,
"output:s"=>\$output,
"methcg:s"=>\$methcg,
"methchg:s"=>\$methchg,
"methchh:s"=>\$methchh,
"minhetfreq:f"=>\$minhetfreq,
"minhomfreq:f"=>\$minhomfreq,
"minquali:i"=>\$minquali,
"mincover:i"=>\$mincover,
"maxcover:i"=>\$maxcover,
"minread2:i"=>\$minread2,
"errorate:f"=>\$errorate,
"mapvalue:i"=>\$mapvalue,
"help"=>\$Help
);
die `pod2text $0` if $Help or not defined $bam or not defined $fasta;
$minhetfreq=0.1 if not defined $minhetfreq;
$minhomfreq=0.85 if not defined $minhomfreq;
$minquali=15 if not defined $minquali;
$mincover=10 if not defined $mincover;
$minread2=2 if not defined $minread2;
$maxcover=1000 if not defined $maxcover;
$errorate=0.02 if not defined $errorate;
$mapvalue=20 if not defined $mapvalue;
#$pvalue ||=0.01;
$output = 'result' if not defined $output;
$methcg = 'meth.cg' if not defined $methcg;
$methchg = 'meth.chg' if not defined $methchg;
$methchh = 'meth.chh' if not defined $methchh;
my $getFlagf = sub {return 1};
if (defined $varOnly) {
$getFlagf = sub {return 0};
}
my %theRegion;
if (defined $regFile) {
my ($cntL,$cntP) = (0,0);
open R,'<',$regFile or die $!;
while (<R>) {
chomp;
my ($chr,$begin,$end)=split /\s+/;
next unless defined $end;
++$cntL;
for my $i ($begin .. $end) {
$theRegion{$chr}{$i}=1;
++$cntP;
}
}
close R;
warn "[!]Regions Load: $cntL lines of $cntP points.\n";
$getFlagf = sub($$) {
if (exists $theRegion{$_[0]} and exists $theRegion{$_[0]}{$_[1]}) {
return 1;
} else {
return 0;
}
}
}
### Example ###
my $chr='1'; my $pos=22345;
my $flag = &{$getFlagf}($chr,$pos);
warn "FLAG: $flag\n";
######
my $eee=2.7;
#$interval = $fasta . ".len";
#if(!(-e $interval)) {
# system("$Bin/chrLenExtract $fasta");
#}
if(system("$Bin/rrbsSnp $fasta $bam $output $methcg $methchg $methchh $minquali $mincover $maxcover $minhetfreq $errorate $mapvalue") != 0) {
die "Error!";
}
#if(system("$Bin/rrbsSnp $interval $fasta $bam $output $methcg $methchg $methchh $minquali $mincover $maxcover $minhetfreq $errorate $mapvalue") != 0) {
# die "Error!";
#}
#
#
my $year_month_day=strftime("%Y%m%d",localtime());
print "##fileformat=VCFv4.3
##fileDate= $year_month_day
##bssnperVersion=1.1
##bssnperCommand=--fa $fasta --input $bam --output $output --methcg $methcg --methchg $methchg --methchh $methchh --minhetfreq $minhetfreq --minhomfreq $minhomfreq --minquali $minquali --mincover $mincover --maxcover $maxcover --minread2 $minread2 --errorate $errorate --mapvalue $mapvalue
##reference=file://$fasta
##Bisulfite=directional>
";
open INTV,"$Bin/samtools-0.1.19/samtools view -H $bam|" or die $!;
#@SQ SN:chr3 LN:198295559
while(<INTV>){
chomp;
my @a=split;
next unless(/SN\:(\S+)/);
my ($chr,$length);
if(/SN\:(\S+)/){
$chr=$1;
}
if(/LN\:(\d+)/){
$length=$1;
}
print "##contig=<ID=$chr,length=$length>\n";
}
close INTV;
print "##ALT=<ID=*,Description=\"Represents allele(s) other than observed.\">
##INFO=<ID=INDEL,Number=0,Type=Flag,Description=\"Indicates that the variant is an INDEL.\">
##INFO=<ID=IDV,Number=1,Type=Integer,Description=\"Maximum number of reads supporting an indel\">
##INFO=<ID=IMF,Number=1,Type=Float,Description=\"Maximum fraction of reads supporting an indel\">
##INFO=<ID=DP,Number=1,Type=Integer,Description=\"Raw read depth\">
##INFO=<ID=AD,Number=R,Type=Integer,Description=\"Total allelic depths\">
##INFO=<ID=ADF,Number=R,Type=Integer,Description=\"Total allelic depths on the forward strand\">
##INFO=<ID=ADR,Number=R,Type=Integer,Description=\"Total allelic depths on the reverse strand\">
##INFO=<ID=MQ0F,Number=1,Type=Float,Description=\"Fraction of MQ0 reads (smaller is better)\">
##FILTER=<ID=Low,Description=\"Low Quality\">
##FORMAT=<ID=GT,Number=1,Type=String,Description=\"Genotype\">
##INFO=<ID=AC,Number=A,Type=Integer,Description=\"Allele count in genotypes for each ALT allele, in the same order as listed\">
##INFO=<ID=AN,Number=1,Type=Integer,Description=\"Total number of alleles in called genotypes\">
##INFO=<ID=DP4,Number=4,Type=Integer,Description=\"Number of high-quality ref-forward , ref-reverse, alt-forward and alt-reverse bases\">
##INFO=<ID=MQ,Number=1,Type=Integer,Description=\"Average mapping quality\">
##FORMAT=<ID=PL,Number=G,Type=Integer,Description=\"List of Phred-scaled genotype likelihoods\">
##FORMAT=<ID=DP,Number=1,Type=Integer,Description=\"Number of high-quality bases\">
##FORMAT=<ID=SP,Number=1,Type=Integer,Description=\"Phred-scaled strand bias P-value\">
##FORMAT=<ID=AD,Number=R,Type=Integer,Description=\"Allelic depths\">
##FORMAT=<ID=ADF,Number=R,Type=Integer,Description=\"Allelic depths on the forward strand\">
##FORMAT=<ID=ADR,Number=R,Type=Integer,Description=\"Allelic depths on the reverse strand\">
##FORMAT=<ID=BSD,Number=8,Type=Integer,Description=\"Depth, ATCG in watson strand and crick strand\">
##FORMAT=<ID=BSQ,Number=8,Type=Integer,Description=\"Avarage Base Quality, ATCG in watson strand and crick strand\">
##FORMAT=<ID=ALFR,Number=R,Type=Float,Description=\"Allele frequency\">
";
#print "#CHROM\tPOS\tID\tREF\tALT\tQUAL\tFILTER\tGENOTYPE\tFREQUENCY\tNumber_of_watson[A,T,C,G]\tNumber_of_crick[A,T,C,G]\tMean_Quality_of_Watson[A,T,C,G]\tMean_Quality_of_Crick[A,T,C,G]\n";
#chr1 10583 G 70,0,0,24 0,2,0,243 33,0,0,32 0,35,0,33
print "#CHROM\tPOS\tID\tREF\tALT\tQUAL\tFILTER\tINFO\tFORMAT\t$bam\n";
open SNP,$output or die "no snp file\n";
while(<SNP>){
next unless(/\w/);
next if(/^#/);
my @a=split;
my $geno=&genotype(\@a);
}
#print "SNP finished\n";
#chr1 10583 G 70,0,0,24 0,2,0,243 33,0,0,32 0,35,0,33
#$minfreq,$minquali,$minread2,$mincover,$pvalue,$interval
#chr1 10006 . C T,A,G 1000 PASS DP=2;ADF=0,1;ADR=0,1;AD=0,2; GT:DP:ADF:ADR:AD:BSD:BSQ 0/1:6:0,0,0,0:54,10,1,1:54,10,1,1:0,0,0,0,1,54,10,1:0,0,0,0,37,36,38,22 0/2:6:2,0,10,0:0,0,0,0:2,0,10,0:0,0,0,0,1,54,10,1:0,0,0,0,37,36,38,22
#chr1 10105 . A C 27 Low DP=2;ADF=0,1;ADR=0,1;AD=0,2; GT:DP:ADF:ADR:AD:BSD:BSQ 0/1:6:2,0:2,2:4,2:0,0,0,0,69,9,0,0:0,0,0,0,36,32,0,0 0/1:6:2,0:2,2:4,2:0,0,0,0,1,54,10,1:0,0,0,0,37,36,38,22
#chr1 10006 . C T 1000 PASS CT 0.152 0,0,0,0 1,10,54,1 0,0,0,0 37,38,36,22
sub genotype
{
my $line=shift;
my @lines=@{$line};
my $genoreturn=&Bayes($line);
my @bayes=split /\t/,$genoreturn;
my $genoqual=$bayes[1];
my $genotypemaybe=$bayes[0];
my @watson=split /\,/,$lines[3];
my @crick=split /\,/,$lines[4];
my @wsq=split /\,/,$lines[5];
my @crq=split /\,/,$lines[6];
#print "$lines[0]\t$lines[1]\t$lines[2]\t$genotypemaybe\n";
my $totaldepth=$watson[0]+$watson[1]+$watson[2]+$watson[3]+$crick[0]+$crick[1]+$crick[2]+$crick[3];
if($genotypemaybe eq "AA"){#genotypeis AA
if($lines[2] =~/A/i){
return "REF";
}
if($lines[2] =~/T/i){#T>AA
my $qvalue=($wsq[0]>$crq[0])?$wsq[0]:$crq[0];
my $depth=$watson[0]+$crick[0]+$watson[1]+$crick[1];
my $var=$watson[0]+$crick[0];
my $adf="$watson[1]\,$watson[0]";
my $adr="$crick[1]\,$crick[0]";
my $ad0=$watson[1]+$crick[1];
my $ad1=$watson[0]+$crick[0];
my $ad="$ad0\,$ad1";
my $ref=sprintf("%.3f",$ad0/$totaldepth);
my $T2A=sprintf("%.3f",$var/$totaldepth);
if($depth >= $mincover && $qvalue >= $minquali && $var >=$minread2 ){
#my $T2A=sprintf("%.3f",$var/$totaldepth);
#my $T=sprintf("%.3f",$ad0/$totaldepth);
if($T2A>=$minhomfreq){
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA\t$genoqual\tPASS\tAA\t$T2A\t".join("\t",@lines[3..6])."\n";
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA\t$genoqual\tPASS\tDP=$totaldepth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$T2A\n";
}else{
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA\t$genoqual\tLow\tAA\t$T2A\t".join("\t",@lines[3..6])."\n";
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA\t$genoqual\tLow\tDP=$totaldepth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$T2A\n";
}
}else{
my $T2A=sprintf("%.3f",$var/$totaldepth);
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA\t$genoqual\tLow\tAA\t$T2A\t".join("\t",@lines[3..6])."\n";
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA\t$genoqual\tLow\tDP=$totaldepth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$T2A\n";
}
}
if($lines[2] =~/C/i){#C>AA
my $qvalue=($wsq[0]>$crq[0])?$wsq[0]:$crq[0];
my $depth=$watson[2]+$crick[2]+$watson[0]+$crick[0];
my $var=$watson[0]+$crick[0];
my $adf="$watson[2]\,$watson[0]";
my $adr="$crick[2]\,$crick[0]";
my $ad0=$watson[2]+$crick[2];
my $ad1=$watson[0]+$crick[0];
my $ad="$ad0\,$ad1";
my $ref=sprintf("%.3f",$ad0/$totaldepth);
if($depth >= $mincover && $qvalue >= $minquali && $var >=$minread2 ){
my $C2A=sprintf("%.3f",$var/$totaldepth);
if($C2A>=$minhomfreq){
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA\t$genoqual\tPASS\tAA\t$C2A\t".join("\t",@lines[3..6])."\n";
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA\t$genoqual\tPASS\tDP=$totaldepth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$C2A\n";
}else{
# print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA\t$genoqual\tLow\tAA\t$C2A\t".join("\t",@lines[3..6])."\n";
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA\t$genoqual\tLow\tDP=$totaldepth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$C2A\n";
}
}else{
my $C2A=sprintf("%.3f",$var/$totaldepth);
# print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA\t$genoqual\tLow\tAA\t$C2A\t".join("\t",@lines[3..6])."\n";
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA\t$genoqual\tLow\tDP=$totaldepth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$C2A\n";
}
}
if($lines[2] =~/G/i){##G>AA
my $qvalue=$wsq[0];
my $depth=$watson[3]+$watson[0]+$crick[0]+$crick[3];
my $var=$watson[0]+$crick[0];
my $G2A;
my $adf="$watson[3]\,$watson[0]";
my $adr="$crick[3]\,$crick[0]";
my $ad0=$watson[3]+$crick[3];
my $ad1=$watson[0]+$crick[0];
my $ad="$ad0\,$ad1";
my $ref;
if($depth>0){
$G2A=sprintf("%.3f",$var/$depth);
$ref=sprintf("%.3f",$ad0/$depth);
}else{
$G2A=0;
}
if($depth >= $mincover && $qvalue >= $minquali && $var >=$minread2 ){
if($G2A>=$minhomfreq){
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA\t$genoqual\tPASS\tAA\t$G2A\t".join("\t",@lines[3..6])."\n";
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA\t$genoqual\tPASS\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$G2A\n";
}else{
# print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA\t$genoqual\tLow\tAA\t".join("\t",@lines[3..6])."\n";
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA\t$genoqual\tLow\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$G2A\n";
}
}else{
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA\t$genoqual\tLow\tAA\t$G2A\t".join("\t",@lines[3..6])."\n";
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA\t$genoqual\tLow\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$G2A\n"; }
}
}
#genotype is AT
if($genotypemaybe eq "AT"){ #genotype is AT
if($lines[2] =~/A/i){#A>AT
my $qvalue=($wsq[1]>$crq[1])?$wsq[1]:$crq[1];;
my $depth=$watson[0]+$watson[1]+$crick[0]+$crick[1];
my $var=$watson[1]+$crick[1];
my $adf="$watson[0]\,$watson[1]";
my $adr="$crick[0]\,$crick[1]";
my $ad0=$watson[0]+$crick[0];
my $ad1=$watson[1]+$crick[1];
my $ad="$ad0\,$ad1";
my $ref=sprintf("%.3f",$ad0/$totaldepth);
if($depth >= $mincover && $qvalue >= $minquali && $var >=$minread2 ){
my $A2T=sprintf("%.3f",$var/$totaldepth);
if($A2T>=$minhetfreq){
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tT\t$genoqual\tPASS\tAT\t$A2T\t".join("\t",@lines[3..6])."\n";
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tT\t$genoqual\tPASS\tDP=$totaldepth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t0/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$A2T\n";
}else{
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tT\t$genoqual\tLow\tDP=$totaldepth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t0/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$A2T\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tT\t$genoqual\tLow\tAT\t$A2T\t".join("\t",@lines[3..6])."\n";
}
}else{
my $A2T=sprintf("%.3f",$var/$totaldepth);
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tT\t$genoqual\tLow\tDP=$totaldepth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t0/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$A2T\n";
# print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tT\t$genoqual\tLow\tAT\t$A2T\t".join("\t",@lines[3..6])."\n";
}
}
if($lines[2] =~/T/i){#T>AT
my $qvalue=($wsq[0]>$crq[0])?$wsq[0]:$crq[0];
my $depth=$watson[0]+$watson[1]+$crick[0]+$crick[1];
my $var=$crick[0]+$watson[0];
my $adf="$watson[1]\,$watson[0]";
my $adr="$crick[1]\,$crick[0]";
my $ad0=$watson[1]+$crick[1];
my $ad1=$watson[0]+$crick[0];
my $ad="$ad0\,$ad1";
my $ref=sprintf("%.3f",$ad0/$totaldepth);
if($depth >= $mincover && $qvalue >= $minquali && $var >=$minread2 ){
my $T2A=sprintf("%.3f",$var/$totaldepth);
if($T2A>=$minhetfreq){
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA\t$genoqual\tPASS\tDP=$totaldepth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t0/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$T2A\n";
# print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA\t$genoqual\tPASS\tAT\t$T2A\t".join("\t",@lines[3..6])."\n";
}else{
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA\t$genoqual\tLow\tDP=$totaldepth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t0/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$T2A\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA\t$genoqual\tLow\tAT\t$T2A\t".join("\t",@lines[3..6])."\n";
}
}else{
my $T2A=sprintf("%.3f",$var/$totaldepth);
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA\t$genoqual\tLow\tAT\t$T2A\t".join("\t",@lines[3..6])."\n";
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA\t$genoqual\tLow\tDP=$totaldepth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t0/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$T2A\n";
}
}
if($lines[2] =~/C/i){#C>AT #add the watson in the new version ##294
my $qvalueA=($wsq[0]>$crq[0])?$wsq[0]:$crq[0];
my $qvalueT=$crq[1];
my $varA=$watson[0]+$crick[0];
my $varT=$crick[1]+$watson[1];
my $depth=$watson[0]+$crick[0]+$crick[1]+$watson[1];
my $adf="$watson[2]\,$watson[0]\,$watson[1]";
my $adr="$crick[2]\,$crick[0]\,$crick[1]";
my $ad0=$watson[2]+$crick[2];
my $ad1=$watson[0]+$crick[0];
my $ad2=$watson[1]+$crick[1];
my $ad="$ad0\,$ad1\,$ad2";
my $ref=sprintf("%.3f",$ad0/$totaldepth);
if($depth >= $mincover && $qvalueA >= $minquali && $qvalueT>=$minquali && $varA >=$minread2 && $varT>=$minread2 ){
my $C2A=sprintf("%.3f",$varA/$totaldepth);
my $C2T=sprintf("%.3f",$varT/$totaldepth);
if($C2A>=$minhetfreq && $C2T>=$minhetfreq){
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA,T\t$genoqual\tPASS\tDP=$totaldepth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/2:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$C2A\,$C2T\n";
}else{
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA,T\t$genoqual\tLow\tAT\t$C2A\,$C2T\t".join("\t",@lines[3..6])."\n";
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA,T\t$genoqual\tLow\tDP=$totaldepth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/2:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$C2A\,$C2T\n";
}
}else{
my $C2A=sprintf("%.3f",$varT/$totaldepth);
my $C2T=sprintf("%.3f",$varT/$totaldepth);
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tAT\t$genoqual\tLow\tAT\t$C2A\,$C2T\t".join("\t",@lines[3..6])."\n";
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA,T\t$genoqual\tLow\tDP=$totaldepth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/2:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$C2A\,$C2T\n";
}
}
if($lines[2] =~/G/i){#G>AT #add the crick in the new version ##326
my $qvalueA=$wsq[0];
my $qvalueT=($wsq[1]>$crq[1])?$wsq[1]:$crq[1];
my $varA=$watson[0]+$crick[0];
my $varT=$crick[1]+$watson[1];
my $depth=$watson[0]+$crick[0]+$crick[1]+$watson[1];
my $adf="$watson[3]\,$watson[0]\,$watson[1]";
my $adr="$crick[3]\,$crick[0]\,$crick[1]";
my $ad0=$watson[3]+$crick[3];
my $ad1=$watson[0]+$crick[0];
my $ad2=$watson[1]+$crick[1];
my $ad="$ad0\,$ad1\,$ad2";
my $ref=sprintf("%.3f",$ad0/$totaldepth);
if($depth >= $mincover && $qvalueA >= $minquali && $qvalueT>=$minquali && $varA >=$minread2 && $varT>=$minread2 ){
my $G2A=sprintf("%.3f",$varA/$totaldepth);
my $G2T=sprintf("%.3f",$varT/$totaldepth);
if($G2A>=$minhetfreq && $G2T>=$minhetfreq){
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA,T\t$genoqual\tPASS\tDP=$totaldepth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/2:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$G2A\,$G2T\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tAT\t$genoqual\tPASS\tAT\t$G2A\,$G2T\t".join("\t",@lines[3..6])."\n";
}else{
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA,T\t$genoqual\tLow\tDP=$totaldepth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/2:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$G2A\,$G2T\n";
# print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tAT\t$genoqual\tLow\tAT\t$G2A\,$G2T\t".join("\t",@lines[3..6])."\n";
}
}else{
my $G2A=sprintf("%.3f",$varA/$totaldepth);
my $G2T=sprintf("%.3f",$varT/$totaldepth);
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA,T\t$genoqual\tLow\tDP=$totaldepth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/2:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$G2A\,$G2T\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tAT\t$genoqual\tLow\tAT\t$G2A\,$G2T\t".join("\t",@lines[3..6])."\n";
}
}
}
#AC #363
if($genotypemaybe eq "AC"){ #genotype is AC # add watson T
if($lines[2] =~/A/i){ #A>AC
my $qvalueC=($wsq[2]>$crq[2])?$wsq[2]:$crq[2];
my $varC=$watson[2]+$crick[2]+$watson[1];
my $depth=$watson[0]+$crick[2]+$watson[2]+$crick[0];
my $watsonC=$watson[1]+$watson[2];
my $adf="$watson[0]\,$watsonC";
my $adr="$crick[0]\,$crick[2]";
my $ad0=$watson[0]+$crick[0];
my $ad1=$watson[2]+$crick[2]+$watson[1];
my $ad="$ad0\,$ad1";
my $ref=sprintf("%.3f",$ad0/$totaldepth);
my $A2C=sprintf("%.3f",$varC/$totaldepth);
if($depth >= $mincover && $qvalueC >= $minquali && $varC >=$minread2 ){
if($A2C>=$minhetfreq){
# print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tC\t$genoqual\tPASS\tAC\t$A2C\t".join("\t",@lines[3..6])."\n";
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tC\t$genoqual\tPASS\tDP=$totaldepth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t0/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$A2C\n";
}else{
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tC\t$genoqual\tLow\tDP=$totaldepth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t0/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$A2C\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tC\t$genoqual\tLow\tAC\t$A2C\t".join("\t",@lines[3..6])."\n";
}
}else{
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tC\t$genoqual\tLow\tDP=$totaldepth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t0/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$A2C\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tC\t$genoqual\tLow\tAC\t$A2C\t".join("\t",@lines[3..6])."\n";
}
}
if($lines[2] =~/T/i){#T>AC ############modify totaldepth 390
my $qvalueA=($wsq[0]>$crq[0])?$wsq[0]:$crq[0];
my $qvalueC=($wsq[2]>$crq[2])?$wsq[2]:$crq[2];
my $varA=$watson[0]+$crick[0];
my $varC=$crick[2]+$watson[2]+$watson[1];
my $depth=$watson[0]+$crick[2]+$watson[2]+$crick[0];
my $T2A=sprintf("%.3f",$varA/$totaldepth);
my $T2C=sprintf("%.3f",$varC/$totaldepth);
my $watsonC=$watson[1]+$watson[2];
my $adf="$watson[1]\,$watson[0]\,$watsonC";
my $adr="$crick[1]\,$crick[0]\,$crick[2]";
my $ad0=$crick[1];
my $ad1=$watson[0]+$crick[0];
my $ad2=$watsonC+$crick[2];
my $ad="$ad0\,$ad1\,$ad2";
my $ref=sprintf("%.3f",$ad0/$totaldepth);
if($depth >= $mincover && $qvalueA >= $minquali && $qvalueC>=$minquali && $varA >=$minread2 && $varC>=$minread2 ){
if($T2A>=$minhetfreq && $T2C>=$minhetfreq){
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA,C\t$genoqual\tPASS\tDP=$totaldepth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/2:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$T2A\,$T2C\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tAC\t$genoqual\tPASS\tAC\t$T2A\,$T2C\t".join("\t",@lines[3..6])."\n";
}else{
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA,C\t$genoqual\tLow\tDP=$totaldepth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/2:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$T2A\,$T2C\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tAC\t$genoqual\tLow\tAC\t$T2A\,$T2C\t".join("\t",@lines[3..6])."\n";
}
}else{
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA,C\t$genoqual\tLow\tDP=$totaldepth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/2:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$T2A\,$T2C\n";
# print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tAC\t$genoqual\tLow\tAC\t$T2A\,$T2C\t".join("\t",@lines[3..6])."\n";
}
}
if($lines[2] =~/C/i){#C>AC 423 add wastonT
my $qvalueA=($wsq[0]>$crq[0])?$wsq[0]:$crq[0];
my $varA=$watson[0]+$crick[0];
my $watsonC=$watson[1]+$watson[2]+$crick[2];
my $depth=$watson[0]+$crick[2]+$watson[2]+$crick[0];
my $adf="$watson[0]\,$watsonC";
my $adr="$crick[0]\,$crick[2]";
my $ad0=$watson[2]+$crick[2]+$watson[1];
my $ad1=$watson[0]+$crick[0];
my $ad="$ad0\,$ad1";
my $ref=sprintf("%.3f",$ad0/$totaldepth);
my $C2A=sprintf("%.3f",$varA/$totaldepth);
if($depth >= $mincover && $qvalueA >= $minquali && $varA >=$minread2 ){
if($C2A>=$minhetfreq){
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA\t$genoqual\tPASS\tDP=$totaldepth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t0/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$C2A\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA\t$genoqual\tPASS\tAC\t$C2A\t".join("\t",@lines[3..6])."\n";
}else{
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA\t$genoqual\tLow\tDP=$totaldepth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t0/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$C2A\n";
# print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA\t$genoqual\tLow\tAC\t$C2A\t".join("\t",@lines[3..6])."\n";
}
}else{
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA\t$genoqual\tLow\tDP=$totaldepth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t0/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$C2A\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA\t$genoqual\tLow\tAC\t$C2A\t".join("\t",@lines[3..6])."\n";
}
}
if($lines[2] =~/G/i){#G>AC 452
my $qvalueA=$wsq[0];
my $qvalueC=($wsq[2]>$crq[2])?$wsq[2]:$crq[2];
my $varA=$watson[0]+$crick[0];
my $varC=$crick[2]+$watson[2]+$watson[1];
my $depth=$watson[0]+$crick[2]+$watson[2]+$crick[0];
my $G2A=sprintf("%.3f",$varA/$totaldepth);
my $G2C=sprintf("%.3f",$varC/$totaldepth);
my $watsonC=$watson[1]+$watson[2];
my $adf="$watson[3]\,$watson[0]\,$watsonC";
my $adr="$crick[3]\,$crick[0]\,$crick[2]";
my $ad0=$crick[3]+$watson[3];
my $ad1=$watson[0]+$crick[0];
my $ad2=$watsonC+$crick[2];
my $ad="$ad0\,$ad1\,$ad2";
my $ref=sprintf("%.3f",$ad0/$totaldepth);
if($depth >= $mincover && $qvalueA >= $minquali && $qvalueC>=$minquali && $varA >=$minread2 && $varC>=$minread2 ){
if($G2A>=$minhetfreq && $G2C>=$minhetfreq){
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA,C\t$genoqual\tPASS\tDP=$totaldepth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/2:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$G2A\,$G2C\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tAC\t$genoqual\tPASS\tAC\t$G2A\,$G2C\t".join("\t",@lines[3..6])."\n";
}else{
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA,C\t$genoqual\tLow\tDP=$totaldepth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/2:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$G2A\,$G2C\n";
# print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tAC\t$genoqual\tLow\tAC\t$G2A\,$G2C\t".join("\t",@lines[3..6])."\n";
}
}else{
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA,C\t$genoqual\tLow\tDP=$totaldepth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/2:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$G2A\,$G2C\n";
# print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tAC\t$genoqual\tLow\tAC\t$G2A\,$G2C\t".join("\t",@lines[3..6])."\n";
}
}
}
#AG genotype
if($genotypemaybe eq "AG"){ #only watson strand
if($lines[2] =~/A/i){#A>AG modify depth 489##
my $qvalueG=($wsq[3]>$crq[3])?$wsq[3]:$crq[3];
my $varG=$watson[3];
my $depth=$watson[0]+$watson[3];##
my $A2G=sprintf("%.3f",$watson[3]/($watson[3]+$watson[0]));
my $adf="$watson[0]\,$watson[3]";
my $adr="0\,0";
my $ad0=$watson[0];
my $ad1=$watson[3];
my $ad="$ad0\,$ad1";
my $ref=sprintf("%.3f",$ad0/($ad0+$ad1));
if($depth >= $mincover && $qvalueG >= $minquali && $varG >=$minread2 ){
if($A2G>=$minhetfreq){
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tG\t$genoqual\tPASS\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t0/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$A2G\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tG\t$genoqual\tPASS\tAG\t$A2G\t".join("\t",@lines[3..6])."\n";
}else{
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tG\t$genoqual\tLow\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t0/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$A2G\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tG\t$genoqual\tLow\tAG\t$A2G\t".join("\t",@lines[3..6])."\n";
}
}else{
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tG\t$genoqual\tLow\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t0/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$A2G\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tG\t$genoqual\tLow\tAC\t$A2G\t".join("\t",@lines[3..6])."\n";
}
}
if($lines[2] =~/T/i){#T>AG 515
my $qvalueA=$wsq[0];
my $qvalueG=($wsq[3]>$crq[3])?$wsq[3]:$crq[3];
my $varA=$watson[0];
my $varG=$watson[3];
my $depth=$watson[0]+$watson[3];
my $T2A=sprintf("%.3f",$varA/$depth);
my $T2G=sprintf("%.3f",$varG/$depth);
my $adf="$watson[1]\,$watson[0]\,$watson[3]";
my $adr="0\,0\,0";
my $ad0=$watson[1]+$crick[1];
my $ad1=$watson[0];
my $ad2=$watson[3];
my $ad="$ad0\,$ad1\,$ad2";
my $ref=sprintf("%.3f",$ad0/$depth);
if($depth >= $mincover && $qvalueA >= $minquali && $qvalueG>=$minquali && $varA >=$minread2 && $varG>=$minread2 ){
if($T2A>=$minhetfreq && $T2G>=$minhetfreq){
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA,G\t$genoqual\tPASS\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/2:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$T2A\,$T2G\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tAG\t$genoqual\tPASS\tAG\t$T2A\,$T2G\t".join("\t",@lines[3..6])."\n";
}else{
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA,G\t$genoqual\tLow\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/2:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$T2A\,$T2G\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tAG\t$genoqual\tLow\tAG\t$T2A\,$T2G\t".join("\t",@lines[3..6])."\n";
}
}else{
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA,G\t$genoqual\tLow\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/2:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$T2A\,$T2G\n";
# print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tAG\t$genoqual\tLow\tAG\t$T2A\,$T2G\t".join("\t",@lines[3..6])."\n";
}
}
if($lines[2] =~/C/i){#C>AG 551
my $qvalueA=$wsq[0];
my $qvalueG=($wsq[3]>$crq[3])?$wsq[3]:$crq[3];
my $varA=$watson[0];
my $varG=$watson[3];
my $depth=$watson[0]+$watson[3];
my $C2A=sprintf("%.3f",$varA/$depth);
my $C2G=sprintf("%.3f",$varG/$depth);
my $adf="$watson[2]\,$watson[0]\,$watson[3]";
my $adr="0\,0\,0";
my $ad0=$watson[1];
my $ad1=$watson[0];
my $ad2=$watson[3];
my $ad="$ad0\,$ad1\,$ad2";
my $ref=sprintf("%.3f",$ad0/$depth);
if($depth >= $mincover && $qvalueA >= $minquali && $qvalueG>=$minquali && $varA >=$minread2 && $varG>=$minread2 ){
if($C2A>=$minhetfreq && $C2G>=$minhetfreq){ #568
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA,G\t$genoqual\tPASS\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/2:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$C2A\,$C2G\n";
# print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tAG\t$genoqual\tPASS\tAG\t$C2A\,$C2G\t".join("\t",@lines[3..6])."\n";
}else{
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA,G\t$genoqual\tLow\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/2:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$C2A\,$C2G\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tAG\t$genoqual\tLow\tAG\t$C2A\,$C2G\t".join("\t",@lines[3..6])."\n";
}
}else{
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA,G\t$genoqual\tLow\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/2:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$C2A\,$C2G\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tAG\t$genoqual\tLow\tAG\t$C2A\,$C2G\t".join("\t",@lines[3..6])."\n";
}
}
if($lines[2] =~/G/i){#G>AG
my $qvalueA=$wsq[0];
my $varA=$watson[0];
my $depth=$watson[3]+$watson[0];
my $G2A=sprintf("%.3f",$varA/$depth);
my $adf="$watson[3]\,$watson[0]";
my $adr="0\,0";
my $ad0=$watson[3];
my $ad1=$watson[0];
my $ad="$ad0\,$ad1";
my $ref=sprintf("%.3f",$ad0/($ad0+$ad1));
if($depth >= $mincover && $qvalueA >= $minquali && $varA >=$minread2 ){
if($G2A>=$minhetfreq){
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA\t$genoqual\tPASS\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t0/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$G2A\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA\t$genoqual\tPASS\tAG\t$G2A\t".join("\t",@lines[3..6])."\n";
}else{
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA\t$genoqual\tLow\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t0/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$G2A\n";
# print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA\t$genoqual\tLow\tAG\t$G2A\t".join("\t",@lines[3..6])."\n";
}
}else{
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA\t$genoqual\tLow\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t0/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$G2A\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tA\t$genoqual\tLow\tAG\t$G2A\t".join("\t",@lines[3..6])."\n";
}
}
}
#TT
if($genotypemaybe eq "TT"){
if($lines[2] =~/A/i){#A>TT 612
my $qvalueT=$crq[1];
my $depth=$watson[0]+$crick[0]+$crick[1]+$watson[1];
my $varT=$crick[1]+$watson[1];
my $adf="$watson[0]\,$watson[1]";
my $adr="$crick[0]\,$crick[1]";
my $ad0=$watson[0]+$crick[0];
my $ad1=$watson[1]+$crick[1];
my $ad="$ad0\,$ad1";
my $ref=sprintf("%.3f",$ad0/($ad0+$ad1));
if($depth >= $mincover && $qvalueT >= $minquali && $varT >=$minread2 ){
my $A2T=sprintf("%.3f",$varT/$depth);
if($A2T>=$minhomfreq){ #627
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tT\t$genoqual\tPASS\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$A2T\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tT\t$genoqual\tPASS\tTT\t$A2T\t".join("\t",@lines[3..6])."\n";
}else{
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tT\t$genoqual\tLow\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$A2T\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tT\t$genoqual\tLow\tTT\t$A2T\t".join("\t",@lines[3..6])."\n";
}
}else{
my $A2T=sprintf("%.3f",$varT/$depth);
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tT\t$genoqual\tLow\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$A2T\n";
# print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tT\t$genoqual\tLow\tTT\t$A2T\t".join("\t",@lines[3..6])."\n";
}
}
if($lines[2] =~/T/i){
return "REF";
}
if($lines[2] =~/C/i){#C>TT
my $qvalueT=$crq[1];
my $depth=$crick[1]+$crick[2]+$watson[1]+$watson[2];
my $varT=$crick[1]+$watson[1];
my $C2T;
if($depth>0){
$C2T=sprintf("%.3f",$varT/$depth);
}else{
$C2T=0;
}
my $adf="$watson[2]\,$watson[1]";
my $adr="$crick[2]\,$crick[1]";
my $ad0=$crick[2]+$watson[2];
my $ad1=$crick[1]+$watson[1];
my $ad="$ad0\,$ad1";
my $ref=sprintf("%.3f",$ad0/($ad0+$ad1));
if($depth >= $mincover && $qvalueT >= $minquali && $varT >=$minread2 ){
if($C2T>=$minhomfreq){#665
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tT\t$genoqual\tPASS\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$C2T\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tT\t$genoqual\tPASS\tTT\t$C2T\t".join("\t",@lines[3..6])."\n";
}else{
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tT\t$genoqual\tLow\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$C2T\n";
# print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tT\t$genoqual\tLow\tTT\t$C2T\t".join("\t",@lines[3..6])."\n";
}
}else{
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tT\t$genoqual\tLow\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$C2T\n";
# print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tT\t$genoqual\tLow\tTT\t$C2T\t".join("\t",@lines[3..6])."\n";
}
}
if($lines[2] =~/G/i){#G>TT
my $qvalueT=$crq[1];
my $depth=$watson[3]+$crick[3]+$crick[1]+$watson[1];
my $varT=$crick[1]+$watson[1];
my $adf="$watson[3]\,$watson[1]";
my $adr="$crick[3]\,$crick[1]";
my $ad0=$crick[3]+$watson[3];
my $ad1=$crick[1]+$watson[1];
my $ad="$ad0\,$ad1";
my $ref=sprintf("%.3f",$ad0/($ad0+$ad1));
if($depth >= $mincover && $qvalueT >= $minquali && $varT >=$minread2 ){
my $G2T=sprintf("%.3f",$varT/$totaldepth);
if($G2T>=$minhomfreq){
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tT\t$genoqual\tPASS\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$G2T\n";
# print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tT\t$genoqual\tPASS\tTT\t$G2T\t".join("\t",@lines[3..6])."\n";
}else{
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tT\t$genoqual\tLow\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$G2T\n";
# print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tT\t$genoqual\tLow\tTT\t$G2T\t".join("\t",@lines[3..6])."\n";
}
}else{
my $G2T=sprintf("%.3f",$varT/$totaldepth);
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tT\t$genoqual\tLow\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$G2T\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tT\t$genoqual\tLow\tTT\t$G2T\t".join("\t",@lines[3..6])."\n";
}
}
}
#CC
if($genotypemaybe eq "CC"){
if($lines[2] =~/A/i){#A>CC
my $qvalueC=($crq[2]>$wsq[2])?$crq[2]:$wsq[2];
my $depth=$watson[0]+$crick[0]+$crick[2]+$watson[2]+$watson[1];
my $varC=$watson[2]+$crick[2]+$watson[1];
my $watsonC=$watson[1]+$watson[2];
my $adf="$watson[0]\,$watsonC";
my $adr="$crick[0]\,$crick[2]";
my $ad0=$crick[0]+$watson[0];
my $ad1=$crick[2]+$watson[2]+$watson[1];
my $ad="$ad0\,$ad1";
my $ref=sprintf("%.3f",$ad0/($ad0+$ad1));
if($depth >= $mincover && $qvalueC >= $minquali && $varC >=$minread2 ){
my $A2C=sprintf("%.3f",$varC/$depth);
if($A2C>=$minhomfreq){
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tC\t$genoqual\tPASS\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$A2C\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tC\t$genoqual\tPASS\tCC\t$A2C\t".join("\t",@lines[3..6])."\n";
}else{
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tC\t$genoqual\tLow\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$A2C\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tC\t$genoqual\tLow\tCC\t$A2C\t".join("\t",@lines[3..6])."\n";
}
}else{
my $A2C=sprintf("%.3f",$varC/$depth);
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tC\t$genoqual\tLow\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$A2C\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tC\t$genoqual\tLow\tCC\t$A2C\t".join("\t",@lines[3..6])."\n";
}
}
if($lines[2] =~/T/i){#T>CC only Crick
my $qvalueC=($crq[2]>$wsq[2])?$crq[2]:$wsq[2];
my $depth=$crick[1]+$crick[2]+$watson[1]+$watson[2];
my $varC=$crick[2]+$watson[2]+$watson[1];
my $watsonC=$watson[2]+$watson[1];
my $adf="0\,$watsonC";
my $adr="$crick[1]\,$crick[2]";
my $ad0=$crick[1]+0;
my $ad1=$crick[2]+$watsonC;
my $ad="$ad0\,$ad1";
my $ref=sprintf("%.3f",$ad0/($ad0+$ad1));
if($depth >= $mincover && $qvalueC >= $minquali && $varC >=$minread2 ){
my $T2C=sprintf("%.3f",$varC/$depth);
if($T2C>=$minhomfreq){
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tC\t$genoqual\tPASS\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$T2C\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tC\t$genoqual\tPASS\tCC\t$T2C\t".join("\t",@lines[3..6])."\n";
}else{
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tC\t$genoqual\tLow\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$T2C\n";
# print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tC\t$genoqual\tLow\tCC\t$T2C\t".join("\t",@lines[3..6])."\n";
}
}else{
my $T2C=sprintf("%.3f",$varC/$depth);
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tC\t$genoqual\tLow\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$T2C\n";
# print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tC\t$genoqual\tLow\tCC\t$T2C\t".join("\t",@lines[3..6])."\n";
}
}
if($lines[2] =~/C/i){#C>CC
return "REF";
}
if($lines[2] =~/G/i){#G>CC ######767
my $qvalueC=($crq[2]>$wsq[2])?$crq[2]:$wsq[2];
my $depth=$watson[3]+$crick[3]+$crick[2]+$watson[2]+$watson[1];
my $varC=$watson[2]+$crick[2]+$watson[1];
my $watsonC=$watson[2]+$watson[1];
my $adf="$watson[3]\,$watsonC";
my $adr="$crick[3]\,$crick[2]";
my $ad0=$crick[3]+$watson[3];
my $ad1=$crick[2]+$watsonC;
my $ad="$ad0\,$ad1";
my $ref=sprintf("%.3f",$ad0/($ad0+$ad1));
if($depth >= $mincover && $qvalueC >= $minquali && $varC >=$minread2 ){
my $G2C=sprintf("%.3f",$varC/$depth);
if($G2C>=$minhomfreq){
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tC\t$genoqual\tPASS\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$G2C\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tC\t$genoqual\tPASS\tCC\t$G2C\t".join("\t",@lines[3..6])."\n";
}else{
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tC\t$genoqual\tLow\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$G2C\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tC\t$genoqual\tLow\tCC\t$G2C\t".join("\t",@lines[3..6])."\n";
}
}else{
my $G2C=sprintf("%.3f",$varC/$depth);
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tC\t$genoqual\tLow\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$G2C\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tC\t$genoqual\tLow\tCC\t$G2C\t".join("\t",@lines[3..6])."\n";
}
}
}
#GG 804 hom add crick strand
if($genotypemaybe eq "GG"){
if($lines[2] =~/A/i){#A>GG hom add crick
my $qvalueG=($crq[3]>$wsq[3])?$crq[3]:$wsq[3];
my $depth=$watson[0]+$watson[3]+$crick[0]+$crick[3];#808
my $varG=$watson[3]+$crick[3]+$crick[0];
my $adf="$watson[0]\,$watson[3]";
my $crickG=$crick[3]+$crick[0];
my $adr="0\,$crickG";
my $ad0=$watson[0]+0;
my $ad1=$watson[3]+$crickG;
my $ad="$ad0\,$ad1";
my $ref=sprintf("%.3f",$ad0/($ad0+$ad1));
if($depth >= $mincover && $qvalueG >= $minquali && $varG >=$minread2 ){
my $A2G=sprintf("%.3f",$varG/$depth);
if($A2G>=$minhomfreq){
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tG\t$genoqual\tPASS\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$A2G\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tG\t$genoqual\tPASS\tGG\t$A2G\t".join("\t",@lines[3..6])."\n";
}else{
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tG\t$genoqual\tLow\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$A2G\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tG\t$genoqual\tLow\tGG\t$A2G\t".join("\t",@lines[3..6])."\n";
}
}else{
my $A2G=sprintf("%.3f",$varG/$depth);
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tG\t$genoqual\tLow\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$A2G\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tG\t$genoqual\tLow\tGG\t$A2G\t".join("\t",@lines[3..6])."\n";
}
}
if($lines[2] =~/T/i){#T>GG 826
my $qvalueG=($crq[3]>$wsq[3])?$crq[3]:$wsq[3];
my $depth=$watson[1]+$watson[3]+$crick[0]+$crick[3]+$crick[1];
my $varG=$watson[3]+$crick[3]+$crick[0];
my $adf="$watson[1]\,$watson[3]";
my $crickG=$crick[3]+$crick[0];
my $adr="$crick[1]\,$crickG";
my $ad0=$watson[1]+$crick[1];
my $ad1=$watson[3]+$crickG;
my $ad="$ad0\,$ad1";
my $ref=sprintf("%.3f",$ad0/($ad0+$ad1));
if($depth >= $mincover && $qvalueG >= $minquali && $varG >=$minread2 ){
my $T2G=sprintf("%.3f",$varG/$depth);
if($T2G>=$minhomfreq){
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tG\t$genoqual\tPASS\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$T2G\n";
# print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tG\t$genoqual\tPASS\tGG\t$T2G\t".join("\t",@lines[3..6])."\n";
}else{
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tG\t$genoqual\tLow\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$T2G\n";
# print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tG\t$genoqual\tLow\tGG\t$T2G\t".join("\t",@lines[3..6])."\n";
}
}else{
my $T2G=sprintf("%.3f",$varG/$depth);
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tG\t$genoqual\tLow\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$T2G\n";
# print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tG\t$genoqual\tLow\tGG\t$T2G\t".join("\t",@lines[3..6])."\n";
}
}
if($lines[2] =~/C/i){#C>GG
my $qvalueG=($crq[3]>$wsq[3])?$crq[3]:$wsq[3];
my $depth=$watson[2]+$watson[3]+$crick[2]+$crick[3]+$crick[1]+$watson[1];
my $varG=$watson[3]+$crick[3]+$crick[0];
my $adf="$watson[2]\,$watson[3]";
my $crickG=$crick[3]+$crick[0];
my $adr="$crick[2]\,$crickG";
my $ad0=$watson[2]+$crick[2];
my $ad1=$watson[3]+$crickG;
my $ad="$ad0\,$ad1";
my $ref=sprintf("%.3f",$ad0/($ad0+$ad1));
if($depth >= $mincover && $qvalueG >= $minquali && $varG >=$minread2 ){
my $C2G=sprintf("%.3f",$varG/$depth);
if($C2G>=$minhomfreq){
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tG\t$genoqual\tPASS\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$C2G\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tG\t$genoqual\tPASS\tGG\t$C2G\t".join("\t",@lines[3..6])."\n";
}else{
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tG\t$genoqual\tLow\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$C2G\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tG\t$genoqual\tLow\tGG\t$C2G\t".join("\t",@lines[3..6])."\n";
}
}else{
my $C2G=sprintf("%.3f",$varG/$depth);
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tG\t$genoqual\tLow\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$C2G\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tG\t$genoqual\tLow\tGG\t$C2G\t".join("\t",@lines[3..6])."\n";
}
}
if($lines[2] =~/G/i){
return "REF";
}
}
#CT 884
if($genotypemaybe eq "CT"){ #only use crick
if($lines[2] =~/A/i){#A>CT
my $qvalueC=($wsq[2]>$crq[2])?$wsq[2]:$crq[2];
my $qvalueT=$crq[1];
my $varC=$crick[2];
my $varT=$crick[1];
my $depth=$varC+$varT+$crick[0]+$watson[0];
my $adf="0\,0\,0";
my $adr="$crick[0]\,$crick[1]\,$crick[2]";
my $ad0=$crick[0];
my $ad1=$crick[1];
my $ad2=$crick[2];
my $ad="$ad0\,$ad1\,$ad2";
my $ref=sprintf("%.3f",$ad0/$depth);
if($depth >= $mincover && $qvalueC >= $minquali && $qvalueT>=$minquali && $varC >=$minread2 && $varT>=$minread2 ){
my $A2C=sprintf("%.3f",$varC/$depth);
my $A2T=sprintf("%.3f",$varT/$depth);
if($A2C>=$minhetfreq && $A2T>=$minhetfreq){
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tT,C\t$genoqual\tPASS\tDP=$totaldepth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/2:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$A2T\,$A2C\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tCT\t$genoqual\tPASS\tCT\t$A2C\,$A2T\t".join("\t",@lines[3..6])."\n";
}else{
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tT,C\t$genoqual\tLow\tDP=$totaldepth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/2:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$A2T\,$A2C\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tCT\t$genoqual\tLow\tCT\t$A2C\,$A2T\t".join("\t",@lines[3..6])."\n";
}
}else{
my $A2C= sprintf("%.3f",$varC/$depth);
my $A2T= sprintf("%.3f",$varT/$depth);
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tT,C\t$genoqual\tLow\tDP=$totaldepth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t1/2:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$A2T\,$A2C\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tCT\t$genoqual\tLow\tCT\t$A2C\,$A2T\t".join("\t",@lines[3..6])."\n";
}
}
if($lines[2] =~/T/i){ #T>CT only crick strand ##########
my $qvalueC=($wsq[2]>$crq[2])?$wsq[2]:$crq[2];
my $varC=$crick[2];
#my $depth=$crick[3]+$watson[3]+$watson[0]+$watson[1]+$crick[1]+$watson[2]+$crick[2];
my $depth=$crick[1]+$crick[2];
my $T2C= sprintf("%.3f",$varC/$depth);
my $adf="0\,0";
my $adr="$crick[1]\,$crick[2]";
my $ad0=$crick[1];
my $ad1=$crick[2];
my $ad="$ad0\,$ad1";
my $ref=sprintf("%.3f",$ad0/($ad0+$ad1));
if($depth >= $mincover && $qvalueC >= $minquali && $varC >=$minread2 ){
if($T2C>=$minhetfreq){
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tC\t$genoqual\tPASS\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t0/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$T2C\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tC\t$genoqual\tPASS\tCT\t$T2C\t".join("\t",@lines[3..6])."\n";
}else{
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tC\t$genoqual\tLow\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t0/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$T2C\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tC\t$genoqual\tLow\tCT\t$T2C\t".join("\t",@lines[3..6])."\n";
}
}else{
print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tC\t$genoqual\tLow\tDP=$depth\;ADF\=$adf\;ADR=$adr\;AD=$ad\;\tGT:DP:ADF:ADR:AD:BSD:BSQ:ALFR\t0/1:$depth:$adf:$adr:$ad:".join("\,",@lines[3,4]).":".join("\,",@lines[5,6]).":$ref\,$T2C\n";
#print "$lines[0]\t$lines[1]\t\.\t$lines[2]\tG\t$genoqual\tLow\tCT\t$T2C\t".join("\t",@lines[3..6])."\n";
}
}
if($lines[2] =~/C/i){ #C>CT
my $qvalueT=$crq[1];