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PopSeq2Geno_CX.pl
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PopSeq2Geno_CX.pl
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#!/usr/bin/perl -w
#Program: Feng Cheng
#Bug report: chengfeng@caas.cn
use strict;
use warnings;
use threads;
use Getopt::Long;
=head1 program discription
Call Variations From Population Samples With High Accuracy
Prerequiried tools for running: BWA, Samtools
=head1 Command-line Option
--inDir [required] The directory where the population resequencing reads are stored.
--outDir [required] The directory for store the filtered reads and other temp file.
--qual [required] File to provide the illumina reads quality
version 1.8 (all >=1.8), or 1.3 (all <1.8).
--ref [required] Fasta format of the reference genome.
--outStat [optional] File to store the statistics during filtering raw reads.
--Gsize [optional] The size of the genome in units of Gb.
Automatic calculated from the reference genome as default.
--folds [optional] How many folds for each sample use to do the calling.
Use all data as default [0].
--build [optional] Build index [1] for the reference, or don't build it [0]
incase it has been built previously. [1].
--pop [optional] The file of polymorphic loci for variant calling.
If not exist, will perform pooled mapping as default [1]
--sam [optional] File to store the samples used in the variant calling.
--pos [optional] File to store the genomic position of variants called.
--geno [optional] File to store the genotypes of variants called.
--help
=head1 Usage
perl popSeq2Geno.pl -inDir example_reads/ -outDir example_reads_temp/ -qual example_quality -ref example_genome.fa -folds 1 -build 1 -pop 1
=cut
my $softpath = "/home/caix/bin";
##--Initialize input data--##
my ($dir,$outDir,$qual,$stat,$gsize,$fold,$refG,$bref,$pop,$sam,$pos,$geno,$help);
GetOptions(
"inDir=s"=>\$dir,
"outDir=s"=>\$outDir,
"qual=s"=>\$qual,
"outStat=s"=>\$stat,
"Gsize=s"=>\$gsize,
"folds=s"=>\$fold,
"ref=s"=>\$refG,
"build=s"=>\$bref,
"pop=s"=>\$pop,
"sam=s"=>\$sam,
"pos=s"=>\$pos,
"geno=s"=>\$geno,
"help"=>\$help,
);
die `pod2text $0` if($help);
die `pod2text $0` if(!defined $dir);
system("rm -rf Reads_temp") if(-e "Reads_temp");
if(! defined $outDir){
$outDir = "Reads_temp/";
}
die `pod2text $0` if(!defined $outDir);
die `pod2text $0` if(!defined $qual);
die `pod2text $0` if(!defined $refG);
if(!defined $stat) {
$stat = "readsFilter.statistics";
}
if(!defined $gsize) {
$gsize = &readGsize($refG);
}
if(!defined $fold) {
$fold = 0;
}
if(!defined $bref) {
$bref = 1;
}
if(!defined $pop) {
$pop = 1;
}
if(!defined $sam) {
$sam = "samples";
}
if(!defined $pos) {
$pos = "positions.loci";
}
if(!defined $geno) {
$geno = "genotypes.loci";
}
#if($pop !~ /^1$/) {
# $fold = 0;
#}
#--Launch the running process-----------------------
&process_main($dir,$outDir,$qual,$stat,$gsize,$fold,$refG,$bref,$pop,$sam,$pos,$geno);
sub process_main {
my ($dir,$outDir,$qual,$stat,$gsize,$fold,$ref,$bref,$pop,$sam,$pos,$geno) = @_;
my (%file,%rfile2rec);
&readFile(\%file,\%rfile2rec,$dir);
if(!(-e $outDir)) {
system("mkdir $outDir");
}
my $fOutDir = $outDir."reads_ft/";
&qualityFilter($qual,\%file,\%rfile2rec,$dir,$fOutDir,$stat);
print "\nAll filtering process run successfully...\n\n";
my %sam2fold;
&readStat($stat,\%sam2fold,$gsize);
my $pfOutDir = $outDir."reads_sel/";
&pickOutReads($fOutDir,$pfOutDir,$fold,\%sam2fold);
if($bref == 1) {
system("$softpath/bwa index $ref"); #--format reference--
system("$softpath/samtools faidx $ref");
}
my $mapDir = $outDir."mapped/";
my $plist;
if($pop =~ /^1$/) {
$plist = "populationPolymorphicLoci.posAlleleT";
&bMapPop($pfOutDir,$mapDir,$ref,$plist,$fold,\%sam2fold);
}
else {
$plist = $pop;
}
my $mapDir2 = $mapDir;
if($fold == 0) {
&bMapIndi($pfOutDir,$mapDir2,$ref,$plist,$pos,$geno,$sam,$fold,\%sam2fold,$pop);
}
else {
$mapDir2 = $outDir."mapped2/";
&bMapIndi($fOutDir,$mapDir2,$ref,$plist,$pos,$geno,$sam,$fold,\%sam2fold,$pop);
}
}
sub readStat {
my ($stat,$sam2fold,$gsize) = @_;
open(my $FR,$stat);
while($_=<$FR>) {
chomp;
my @temp = split(/\t/);
$temp[0] =~ /\/([^\/]+)_1\.fq[s]*\.gz/;
my $sam = $1;
my $rfold = $temp[7]/1000000000/$gsize;
$sam2fold->{$sam} = $rfold;
#print "$sam\t$rfold\n";
}
close($FR);
}
sub pickOutReads {
my ($fOutDir,$pfOutDir,$fold,$sam2fold) = @_;
if(-e $pfOutDir) {
system("rm -rf $pfOutDir");
}
system("mkdir $pfOutDir");
if($fold!=0) {
foreach my $sam (sort {$a cmp $b} keys %$sam2fold) {
my $rr1 = $sam."_1.fq.ft.gz";
my $rr2 = $sam."_2.fq.ft.gz";
my $r1 = $sam."_1.fq.gz";
my $r2 = $sam."_2.fq.gz";
my $in1 = $fOutDir.$rr1; #caix
my $in2 = $fOutDir.$rr2; #caix
my $out1 = $pfOutDir.$r1; #caix
my $out2 = $pfOutDir.$r2; #caix
if($sam2fold->{$sam}>$fold) {
my $nfold = $fold/$sam2fold->{$sam};
open(my $FR1,"gzip -dc $in1 |");
open(my $FR2,"gzip -dc $in2 |");
open(my $FW1,"| gzip >$out1");
open(my $FW2,"| gzip >$out2");
my (@L1,@L2);
while($_=<$FR1>) {
$L1[0] = $_;
$L1[1] = <$FR1>;
$L1[2] = <$FR1>;
$L1[3] = <$FR1>;
$L2[0] = <$FR2>;
$L2[1] = <$FR2>;
$L2[2] = <$FR2>;
$L2[3] = <$FR2>;
if(rand(1)<$nfold) {
print $FW1 $L1[0];
print $FW1 $L1[1];
print $FW1 $L1[2];
print $FW1 $L1[3];
print $FW2 $L2[0];
print $FW2 $L2[1];
print $FW2 $L2[2];
print $FW2 $L2[3];
}
}
close($FR1);
close($FR2);
close($FW1);
close($FW2);
}
else {
system("ln -s ../../$in1 $out1");
system("ln -s ../../$in2 $out2"); #caix
}
}
}
else {
foreach my $sam (sort {$a cmp $b} keys %$sam2fold) {
#my $r1 = $sam."_1.fq.ft.gz";
#my $r2 = $sam."_2.fq.ft.gz";
#my $ls1 = $fOutDir.$r1;
#my $ls2 = $fOutDir.$r2;
#$ls1 =~s/^[^\/]+\///;
#$ls2 =~s/^[^\/]+\///;
#system("ln -s ../$ls1 $pfOutDir");
#system("ln -s ../$ls2 $pfOutDir");
my $rr1 = $sam."_1.fq.ft.gz";
my $rr2 = $sam."_2.fq.ft.gz";
my $r1 = $sam."_1.fq.gz";
my $r2 = $sam."_2.fq.gz";
my $in1 = $fOutDir.$rr1; #caix
my $in2 = $fOutDir.$rr2; #caix
my $out1 = $pfOutDir.$r1;
my $out2 = $pfOutDir.$r2;
system("ln -s ../../$in1 $out1");
system("ln -s ../../$in2 $out2"); #caix
}
}
}
sub readGsize {
my ($in) = @_;
my %sca2seq;
&readFasta($in,\%sca2seq);
my $gsize = 0;
foreach my $sca (keys %sca2seq) {
$gsize += length($sca2seq{$sca});
}
$gsize /= 1000000000;
return $gsize;
}
sub readFasta {
my ($in,$id2seq) = @_;
open(my $SFR,$in);
my $id;
while($_=<$SFR>) {
if(/^>([^\s^\n]+)\s*\n*/) {
$id = $1;
$id2seq->{$id} = "";
}
else {
chomp;
$id2seq->{$id} .= $_;
}
}
close($SFR);
}
sub bMapPop {
my ($dir,$outDir,$ref,$plist,$fold,$sam2fold) = @_;
my (%file,%file2rec);
##print "$dir\n";
&readFile(\%file,\%file2rec,$dir);
&bwaMapPop(\%file,\%file2rec,$dir,$outDir,$ref,$plist,$fold,$sam2fold);
print "\nPopulation mapping process run successfully...\n\n";
&mergeBam($outDir,$plist,$ref,$fold,$sam2fold);
}
sub bwaMapPop {
my ($file,$file2rec,$dir,$outDir,$ref,$plist,$fold,$sam2fold) = @_;
if(-e $outDir) {
system("rm -rf $outDir");
}
system("mkdir $outDir");
my $sample;
my $posF = $plist; #--it depends----
foreach $sample (sort {$a cmp $b} keys %$file) {
print "\nStart processing $sample...\n";
# my $in1 = $sample."_1.fq.ft.gz";
# my $in2 = $sample."_2.fq.ft.gz";
my $in1 = $sample."_1.fq.gz"; #caix
my $in2 = $sample."_2.fq.gz";
#print "$in1\t$in2\n";
if(exists($file2rec->{$in1}) && exists($file2rec->{$in2})) {
my $outSam = $outDir.$sample.".cf.sam";
&bmap($dir,$outDir,$in1,$in2,$outSam,$sample,$ref);
system("rm $outSam");
}
}
}
sub mergeBam {
my ($outDir,$plist,$ref,$fold,$sam2fold) = @_;
my $outDirm = $outDir."*.bam";
my $samID = "merge_mapped";
system("$softpath/samtools merge -@ 6 $samID.bam $outDirm");
my $t1 = $samID.".bam.sorted";
system("$softpath/samtools sort $samID.bam $t1");
#system("rm $samID.bam");
print "bam sorting is OK!\n";
my $t2 = $t1.".bam";
system("$softpath/samtools index $t2");
my @sam = keys %$sam2fold;
my $nsam = @sam;
my $bdep;
if($fold==0) {
foreach my $sam (keys %$sam2fold) {
$bdep += $sam2fold->{$sam};
}
}
else {
foreach my $sam (keys %$sam2fold) {
if($sam2fold->{$sam}<$fold) {
$bdep += $sam2fold->{$sam};
}
else {
$bdep += $fold;
}
}
}
my ($minDep,$maxDep);
#$minDep = $bdep * 0.5;
#if($minDep < 10) {
# $minDep = 10;
#}
$maxDep = $bdep * 2;
$maxDep = $bdep * 4;
#print "$maxDep: $ref: $t2: $samID\n";
system("$softpath/samtools mpileup -q 30 -Q 30 -C50 -d $maxDep -L $maxDep -ugf $ref $t2 | $softpath/bcftools view -p 0.9999 -vcg - > $samID.cons.vcf");
my $vfile = $samID.".cons.vcf";
#print "$vfile\n";
my $minAdep = 3; #--it depends---
&screenGenotypeBwa($vfile,$maxDep,$minAdep);
$minDep = 10; #--it depends---
system("awk '\(\$5!~/,/ && \$5~/\\w/ && \$6+\$7>=$minDep\)' $samID.cons.vcf.genotype > $plist");
#system("rm $t2 $t2.bai");
#system("rm $vfile $samID.cons.vcf.genotype");
}
sub screenGenotypeBwa {
my ($in,$dep,$minAdep) = @_;
my $out = $in.".genotype";
open(my $FR,$in);
open(my $FW,">$out");
##min depth for alleles in total samples is set here!
while($_=<$FR>) {
chomp;
my @temp = split(/\t/);
#if($temp[4]=~/\w/ && !($temp[4]=~/,/)) {
#if(/^[^#]/ && $temp[4]=~/\w/) {
if(/^[^#]/) {
if($temp[7] =~ /;DP4=(\d+),(\d+),(\d+),(\d+);/) {
my ($d1,$d2) = ($1+$2,$3+$4);
my ($ref,$a1,$a2) = ($temp[3],$temp[3],$temp[4]);
if($d1>=$minAdep && $d1<=$dep && $d2>=$minAdep && $d2<=$dep) {
#if($a2 eq $ref) {
# $a2 = $a1;
# $a1 = $ref;
#}
#elsif($a1 ne $ref && $a2 ne $ref) {
# $a1 = "N";
# $a2 = "N";
#}
}
elsif($d1>=$minAdep && $d1<=$dep && $d2==0) {
$a2 = $a1;
$d2 = $d1;
}
elsif($d2>=$minAdep && $d2<=$dep && $d1==0) {
$a1 = $a2;
$d1 = $d2;
}
else {
$a1 = "N";
$a2 = "N";
}
#if(!($a2=~/^\w$/)) {
# $a1 = "N";
# $a2 = "N";
#}
if($a1 ne "N" && $a2 ne "N" && $a1 ne $a2) {
print $FW "$temp[0]\t$temp[1]\t$ref\t$a1\t$a2\t$d1\t$d2\n";
}
}
}
}
close($FR);
close($FW);
}
sub bMapIndi {
my ($dir,$outDir,$ref,$plist,$posiF,$genoF,$samF,$fold,$sam2fold,$pop) = @_;
#if($pop !~ /^1$/) {
# if(-e $outDir) {
# system("rm -rf $outDir");
# }
# system("mkdir $outDir");
#}
my (%file,%file2rec);
&readFile(\%file,\%file2rec,$dir);
&bwaMap(\%file,\%file2rec,$dir,$outDir,$ref,$plist,$posiF,$genoF,$samF,$fold,$sam2fold,$pop);
print "\nAll individual calling process run successfully...\n\n\n";
}
sub bwaMap {
my ($file,$file2rec,$dir,$outDir,$ref,$plist,$posiF,$genoF,$samF,$fold,$sam2fold,$pop) = @_;
if(!(-e $outDir)) {
system("mkdir $outDir");
}
##my ($sample,%i2geno);
my $sample;
my $posF = $plist; #--it depends----
##-snp--
##my (%i2pos,%i2alle);
##&readPos($posF,\%i2pos,\%i2alle);
foreach $sample (sort {$a cmp $b} keys %$file) {
print "\nStart processing $sample...\n";
my ($in1,$in2);
#$in1 = $sample."_1.fq.ft.gz";
#$in2 = $sample."_2.fq.ft.gz";
$in1 = $sample."_1.fq.gz";
$in2 = $sample."_2.fq.gz";
if(exists($file2rec->{$in1}) && exists($file2rec->{$in2})) {
my $outSam = $outDir.$sample.".cf.sam";
if($fold!=0 || $pop!~/^1$/) {
&bmap($dir,$outDir,$in1,$in2,$outSam,$sample,$ref);
}
##&callVar($outSam,$ref,\%i2geno,$posF,\%i2pos,\%i2alle);
&callVar($outSam,$ref,$posF);
}
}
##system("perl extractGP.pl $outDir $posF $posiF $genoF $samF");
print "\nWe are here for outputing genotypes...\n";
&extractGP($outDir,$posF,$posiF,$genoF,$samF,$fold,$sam2fold,);
##print "$outDir\n";
}
sub extractGP {
my ($outDir,$posF,$posiF,$genoF,$samF,$fold,$sam2fold) = @_;
#-snp--
my (%i2pos,%i2alle);
&readPos($posF,\%i2pos,\%i2alle);
my %i2geno;
&extractGeno($outDir,\%i2pos,\%i2alle,\%i2geno,$samF,$sam2fold);
my ($posiF1,$genoF1) = ($posiF.".snp",$genoF.".snp");
&output(\%i2pos,\%i2alle,\%i2geno,$posiF1,$genoF1);
#-indel--
my (%pindex2oldPos,%oldPos2rec,%ppos2indel);
&popIndel($posF,\%pindex2oldPos,\%oldPos2rec,\%ppos2indel);
my %pos2indelG;
&extractIndel($outDir,\%pindex2oldPos,\%oldPos2rec,\%ppos2indel,\%pos2indelG,$samF,$sam2fold);
my ($posiF2,$genoF2) = ($posiF.".indel",$genoF.".indel");
&outputIndel(\%pindex2oldPos,\%oldPos2rec,\%ppos2indel,\%pos2indelG,$posiF2,$genoF2);
}
sub outputIndel {
my ($index2oldpos,$oldPos2rec,$ppos2indel,$pos2geno,$posiF,$genoF) = @_;
open(my $FW1,">$posiF");
open(my $FW2,">$genoF");
my %pt2rec;
foreach my $i (sort {$a <=> $b} keys %$index2oldpos) {
my $opos = $index2oldpos->{$i};
my $pt = $oldPos2rec->{$opos};
if(!exists($pt2rec{$pt})) {
$pt2rec{$pt} = "Y";
if(exists($pos2geno->{$pt})) {
my $geno = $pos2geno->{$pt};
my $tg = $geno;
$tg =~ s/N//g;
if($tg=~/\w/) {
print $FW1 "$ppos2indel->{$pt}\n";
print $FW2 "$geno\n";
}
}
}
}
close($FW1);
close($FW2);
}
sub extractIndel {
my ($outDir,$pindex2oldPos,$oldPos2rec,$ppos2indel,$pos2geno,$samF,$sam2fold) = @_;
open(my $FW,">$samF.indel");
opendir(DIR,$outDir) || die "Error in opening dir $outDir\n";
my $filename;
my $mdep;
while(($filename = readdir(DIR))){
if($filename=~/^(.+)\.cf\.vcf\.list/) {
my $sam = $1;
print $FW "$sam\n";
my $rfold = $sam2fold->{$sam};
if($rfold>3) {
$mdep = 0; #**to be revised***************
}
else {
$mdep = 0;
}
my %pos2rec;
&readIndel($outDir.$filename,\%pos2rec);
my %indel2rec;
my %crtPt2geno = ();
my $index = 1;
foreach my $index (keys %$pindex2oldPos) {
my $opos = $pindex2oldPos->{$index};
my $pt = $oldPos2rec->{$opos};
my $indel = $ppos2indel->{$pt};
if(!exists($crtPt2geno{$pt}) || $crtPt2geno{$pt} ne "GG") {
if(exists($pos2rec{$opos})) {
my $data = $pos2rec{$opos};
if($data =~ /INDEL/) {
my @temp = split(/\t/,$data);
my ($d1,$d2);
if($temp[7]=~/;DP4=(\d+),(\d+),(\d+),(\d+);/) {
($d1,$d2) = ($1+$2,$3+$4);
}
my ($spt,$sindel);
my ($len1,$len2) = (length($temp[3]),length($temp[4]));
&typeIndel($temp[0],$temp[1],$temp[3],$temp[4],$len1,$len2,\$spt,\$sindel);
if($indel eq $sindel) {
if($d1>0 && $d2>0) {
$crtPt2geno{$spt} = "CG";
}
elsif($d2>0) {
$crtPt2geno{$spt} = "GG";
}
elsif($d1>0) {
$crtPt2geno{$spt} = "CC";
}
else {
if(!exists($crtPt2geno{$spt})) {
$crtPt2geno{$spt} = "NN";
}
}
}
else {
if(!exists($crtPt2geno{$pt})) {
$crtPt2geno{$pt} = "NN";
}
}
}
else {
$crtPt2geno{$pt} = "CC";
}
}
else {
if(!exists($crtPt2geno{$pt})) {
$crtPt2geno{$pt} = "NN";
}
}
}
}
my %pt2rec = ();
foreach my $index (keys %$pindex2oldPos) {
my $opos = $pindex2oldPos->{$index};
my $pt = $oldPos2rec->{$opos};
if(!exists($pt2rec{$pt})) {
$pt2rec{$pt} = "Y";
my $cgeno = $crtPt2geno{$pt};
if(exists($pos2geno->{$pt})) {
$pos2geno->{$pt} .= $cgeno;
}
else {
$pos2geno->{$pt} = $cgeno;
}
}
}
}
}
close($FW);
}
sub readIndel {
my ($in,$pos2rec) = @_;
open(my $FR,$in);
while($_=<$FR>) {
if(!(/^#/)) {
chomp;
my @temp = split(/\t/);
if(!($temp[4]=~/,/)) {
my $pos = $temp[0]."\t".$temp[1];
$pos2rec->{$pos} = $_;
}
}
}
close($FR);
}
sub popIndel {
my ($in,$pindex2oldPos,$oldPos2rec,$pos2indel) = @_;
my $count = 1;
my %pt2rec;
open(my $FR,$in);
open(my $FW,">$in.InDel");
while($_=<$FR>) {
chomp;
my @temp = split(/\t/);
my ($pold,$pt,$indel);
my ($len1,$len2) = (length($temp[3]),length($temp[4]));
if($len1>1 || $len2>1) {
&typeIndel($temp[0],$temp[1],$temp[3],$temp[4],$len1,$len2,\$pt,\$indel);
$pold = "$temp[0]\t$temp[1]";
$pindex2oldPos->{$count} = $pold;
$oldPos2rec->{$pold} = $pt;
#$index2pos->{$count} = $pt;
$pos2indel->{$pt} = $indel;
$count += 1;
if(!exists($pt2rec{$pt})) {
print $FW "$indel\t$temp[5]\t$temp[6]\n";
$pt2rec{$pt} = "Y";
}
}
}
#foreach my $i (sort {$a<=>$b} keys %$pindex2oldPos) {
# print "$i\t$pindex2oldPos->{$i}\t$oldPos2rec->{$pindex2oldPos->{$i}}\n";
#}
close($FR);
close($FW);
}
sub typeIndel {
my ($chr,$pos,$a1,$a2,$len1,$len2,$pt,$indel) = @_;
my ($type,$size,$length,$allel);
$length = abs($len1-$len2);
$size = $length;
if($len2<$len1) {
$type = "D";
$pos += $len2;
$a1 =~ s/^(\w){$len2}//;
$allel = $a1;
}
else {
$type = "I";
$pos += $len1;
$a2 =~ s/^(\w){$len1}//;
$allel = $a2;
}
$$pt = "$chr\t$pos";
$$indel = "$$pt\t$type$size\t$allel";
}
sub output {
my ($i2pos,$i2alle,$i2geno,$posiF,$genoF) = @_;
my ($out1,$out2) = ($posiF,$genoF); #--it depends--
open(my $FW1,">$out1");
open(my $FW2,">$out2");
my $count = 0;
foreach my $i (sort {$a <=> $b} keys %$i2pos) {
my $alle = $i2alle->{$i};
my @ta = split(/:/,$alle);
my $tg = $i2geno->{$i};
$tg =~ s/N//g;
if($tg=~/\w/) {
$tg =~ s/$ta[0]//g;
$tg =~ s/$ta[1]//g;
if(!($tg=~/\w/)) {
my $pos = $i2pos->{$i};
$pos =~ s/-/\t/;
print $FW1 "$pos\t$ta[0]\t$ta[1]\n";
print $FW2 "$i2geno->{$i}\n";
}
}
else {
$count += 1;
}
}
#print "$count\n";
close($FW1);
close($FW2);
}
sub extractGeno {
my ($outDir,$i2pos,$i2alle,$i2geno,$samF,$sam2fold) = @_;
##my $gfile; #.list;
open(my $FW,">$samF.snp");
opendir(DIR,$outDir) || die "Error in opening dir $outDir\n";
my $filename;
my $mdep;
while(($filename = readdir(DIR))){
if($filename=~/^(.+)\.cf\.vcf\.list/) {
my $sam = $1;
print $FW "$sam\n";
my $rfold = $sam2fold->{$sam};
if($rfold>3) {
$mdep = 0; #**to be revised***************
}
else {
$mdep = 0;
}
my %pos2rec;
&readData($outDir.$filename,\%pos2rec);
foreach my $i (sort {$a <=> $b} keys %$i2pos) {
my $pos = $i2pos->{$i};
my @sa;
($sa[0],$sa[1]) = ("N","N");
if(!exists($pos2rec{$pos})) {
if(!exists($i2geno->{$i})) {
$i2geno->{$i} = "NN";
}
else {
$i2geno->{$i} .= "NN";
}
}
else {
my $data = $pos2rec{$pos};
my @temp = split(/\t/,$data);
if($temp[7]=~/;DP4=(\d+),(\d+),(\d+),(\d+);/) {
my ($d1,$d2) = ($1+$2,$3+$4);
if($d1>$mdep && $d2>$mdep) {
if($temp[3]=~/^\w$/ && $temp[4]=~/^\w$/) {
$sa[0] = $temp[3];
$sa[1] = $temp[4];
}
}
elsif($d1>$mdep) {
if($temp[3]=~/^\w$/ && $temp[4]=~/^.{1}$/) {
$sa[0] = $temp[3];
$sa[1] = $temp[3];
}
}
elsif($d2>$mdep) {
if($temp[3]=~/^\w$/ && $temp[4]=~/^\w$/) {
$sa[0] = $temp[4];
$sa[1] = $temp[4];
}
}
}
if(!exists($i2geno->{$i})) {
$i2geno->{$i} = "$sa[0]$sa[1]";
}
else {
$i2geno->{$i} .= "$sa[0]$sa[1]";
}
}
}
}
}
close($FW);
closedir(DIR);
}
sub readData {
my ($gfile,$pos2rec) = @_;
open(my $FR,$gfile);
my $index = 1;
while($_=<$FR>) {
if(!(/^#/) && !(/INDEL/)) {
chomp;
my @temp = split(/\t/);
if($temp[3]=~/^\w$/ && $temp[4]=~/^[\.\w]$/) {
my $pos = $temp[0]."-".$temp[1];
$pos2rec->{$pos} = $_;
}
}
}
close($FR);
}
sub readPos {
my ($posF,$i2pos,$i2alle) = @_;
open(my $FR,$posF);
open(my $FW,">$posF.SNP");
my $index = 1;
my %pt2rec;
while($_=<$FR>) {
if(!(/^#/)) {
chomp;
my @temp = split(/\t/);
if($temp[3]=~/^\w$/ && $temp[4]=~/^\w$/) {
$i2pos->{$index} = $temp[0]."-".$temp[1];
$i2alle->{$index} = $temp[3].":".$temp[4];
$index += 1;
my $pt = "$temp[0]\t$temp[1]";
if(!exists($pt2rec{$pt})) {
print $FW "$temp[0]\t$temp[1]\t$temp[3]\t$temp[4]\t$temp[5]\t$temp[6]\n";
$pt2rec{$pt} = "Y";
}
}
}
}
close($FR);
close($FW);
}
sub callVar {
my ($outSam,$ref,$posF) = @_;
$outSam =~ /^(.+)\.sam$/;
my $sample = $1;
system("rm $outSam");
my $t1 = $sample.".bam";
my $t2 = $t1.".sorted";
system("$softpath/samtools sort -@ 20 $t1 $t2");
system("rm $t1");
my $t3 = $t1.".sorted.bam";
system("$softpath/samtools index $t3");
my $t4 = $sample.".vcf";
system("$softpath/samtools mpileup -q 20 -Q 30 -C50 -l $posF -ugf $ref $t3 | $softpath/bcftools view -p 0.99 -cg - > $t4.list");
##system("rm $t3 $t3.bai");
print "$sample calling is clear now...\n";
}