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bam_exclude_reads_by_windows_size_and_numpairs.pl
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#!/usr/bin/env perl
use strict;
use warnings;
use List::Util qw/sum min max/;
use Data::Dumper qw/Dumper/;
use constant USAGE =><<EOH;
usage: $0 bam.stats fasta.fai window_size1 window_size2 num_pairs output_prefix
v20170228
Requirements
List::Util; Data::Dumper
Input and Output:
bam.stats: bam_scaffolding.stats_noBioDBSAM.pl output_prefix
fasta.fai: fasta index
length/pairs: how many pairs in such a length for a scaffolding
output: readname.output .oneonly .window.pairs .filterout
Examples:
bam_exclude_reads_by_windows_size_and_numpairs.pl ../MP40Kb7lib.subseq.R1R2.st.merge.final.bam.stats ../../ta3bAllScaffoldsV443.genom.fa.fai 10000 5 testout
# Window distance
cut -f 11,13 testout.distance | perl -ne 'chomp; \@arr=split(/\t/);next unless (scalar(\@arr)==2 and \$arr[0] =~/^\\d+\$/); print \$arr[0], "\\t", \$arr[1],"\\n";' > log
# strand
grep -v ^'#' testout.distance | cut -f 2,5,8 | sort -u > log
perl -ne 'chomp; \@arr=split(/\\t/);if (exists \$hash{\$arr[0]} and \$hash{\$arr[0]}{\$arr[1]}) {die "Error: repeat: \$_ \\n";} if (exists \$hash{\$arr[1]} and \$hash{\$arr[1]}{\$arr[0]}) {next;} \@arr2=split(/\\\//, \$arr[2]); print \$arr2[0], "\\t", \$arr2[1], "\\n"; \$hash{\$arr[0]}{\$arr[1]}++;' log
EOH
die USAGE if (scalar(@ARGV) !=6 or $ARGV[0] eq '-h' or $ARGV[0] eq '--help');
my $bamstats=$ARGV[0];
my $fastaindex=$ARGV[1];
my $windows1=$ARGV[2];
my $windows2=$ARGV[3];
my $numpairs=$ARGV[4];
my $output=$ARGV[5];
### Configurable start ###
my $maxmappingtimes=1;
my $ratiothreshold=5;
my $min_allowed_insert=20000;
my $max_allowed_insert=60000;
### Configurable end ###
die "Error: invalid bam.stats input\n" unless (defined $bamstats and -s $bamstats);
die "Error: invalid fasta.fai input\n" unless (defined $fastaindex and -s $fastaindex);
die "Error: invalid window_size\n" unless (defined $windows1);
if ($windows1=~/^\d+[kK]$/) {
print "Info: window_size in Kilobase accepted\n";
$windows1=~s/[kK]$/000/;
}
elsif ($windows1=~/^\d+[mM]$/) {
print "Info: window_size in megabase accepted\n";
$windows1=~s/[mM]$/000000/;
}
elsif ($windows1=~/^\d+[gG]$/) {
print "Info: window_size in gigabase accepted\n";
$windows1=~s/[gG]$/000000000/;
}
die "Error: invalid window_size1: $windows1\n" unless ($windows1 =~/^\d+$/);
if ($windows2=~/^\d+[kK]$/) {
print "Info: window_size in Kilobase accepted\n";
$windows2=~s/[kK]$/000/;
}
elsif ($windows2=~/^\d+[mM]$/) {
print "Info: window_size in megabase accepted\n";
$windows2=~s/[mM]$/000000/;
}
elsif ($windows2=~/^\d+[gG]$/) {
print "Info: window_size in gigabase accepted\n";
$windows2=~s/[gG]$/000000000/;
}
die "Error: invalid window_size2: $windows2\n" unless ($windows2 =~/^\d+$/);
die "Error: invalid num_pairs [only numbers 0-9 are accepted, integer]\n" unless (defined $numpairs and $numpairs=~/^\d+$/);
die "Error: invalid output\n" unless (defined $output);
unlink "$output.oneonly" if (-e "$output.oneonly");
unlink "$output.window$windows1-$windows2.pairs$numpairs" if (-e "$output.window$windows1-$windows2.pairs$numpairs");
unlink "$output.filterout" if (-e "$output.filterout");
unlink "$output.distance" if (-e "$output.distance");
unlink "$output.overlap" if (-e "$output.overlap");
unlink "$output.scaffold.pairs" if (-e "$output.scaffold.pairs");
unlink "$output.finalreadids" if (-e "$output.finalreadids");
unlink "$output.finalrefsids" if (-e "$output.finalrefsids");
### Default
my %strand=();
my $strness='NaN';
my $ratio='NaN';
my %finalarray=();
my %idhash=();
my $linenum=0;
my %seqlength=();
my $testwindows=0;
my $test_overlap=0;
my @insertsum=();
my $insertnum=0;
my $estimatedinsertsize=0;
my $estimatedgap=0;
my $maxinsert=0;
my %read2gap=();
my $readproblematic=0;
my $printthislink=0;
my %printreads=();
my %printrefs=();
### Read sequence length
open (FASTAINDEX, " < $fastaindex") || die "Error: can not open fasta.fai\n";
while (my $line=<FASTAINDEX>) {
chomp $line;
$linenum++;
my @arr=split(/\t/, $line);
unless ($arr[0]=~/^\S+$/ and $arr[1]=~/^\d+$/) {
die "Error: invalid fasta line: $line\n";
}
if (exists $seqlength{$arr[0]}) {
die "Error: repeated sequence ID: $arr[0]\n";
}
$seqlength{$arr[0]}=$arr[1];
}
close FASTAINDEX;
print "\n##### SUMMARY 1 #####\n";
print " Read total fasta.fai lines: $linenum\n";
print " Total seqeunces: ", scalar(keys %seqlength), "\n\n";
open (BAMSTATSIN, " < $bamstats") || die "Error: can not open bam.stats: $bamstats\n";
open (FILTERED, " > $output.window$windows1-$windows2.pairs$numpairs") || die "Error: can not write filtered reads: $output.window$windows1-$windows2.pairs$numpairs\n";
open (FILTEROUT, " > $output.filterout") || die "Error: can not write filter_out reads: $output.filterout\n";
open (DISTANCE, " > $output.distance") || die "Error: can not write distance reads: $output.distance\n";
print DISTANCE "#No/Total-NumReads\tRef1name\tRef1len\tRef1std\tRef2name\tRef2len\tRef2std\tS/D\tratio\tAssemblyStd\tDist1\tDist1Detail\tDist2\tDist2Detail\tTest_overlap\tMeaninternalSize\tDetail\n";
open (OVERLAP, " > $output.overlap") || die "Error: can not write contig overlap: $output.overlap\n";
open (SCAFPAIR, " > $output.scaffold.pairs") || die "Error: can not write scaffold.pairs: $output.scaffold.pairs\n";
print SCAFPAIR "Scaffold1\tScaffold2\tST/RC\tDirection\tNum_Fit_Grooup\tNum_Group\tPerc_Fit\tOverlap\tMeanDist\tStdevDist\tMeanGap\tStdevGap\n";
open (FINALREADLIST, " > $output.finalreadids") || die "Error: can not write finalreadids: $output.finalreadids\n";
open (FINALREFSLIST, " > $output.finalrefsids") || die "Error: can not write finalrefsids: $output.finalrefsids\n";
$linenum=0;
while (my $line=<BAMSTATSIN>) {
chomp $line;
$linenum++;
my @arr=split(/\t/, $line);
# $arr[0]=quotemeta($arr[0]);
if (exists $idhash{$arr[0]}{$arr[2]}{$arr[1]}) {
$idhash{$arr[0]}{'EXCLUDEDPLZ'}++;
next;
}
if ($arr[5] eq '+') {
$idhash{$arr[0]}{$arr[2]}{$arr[1]}{'pos'}=$arr[3];
$idhash{$arr[0]}{$arr[2]}{$arr[1]}{'str'}=$arr[5];
}
elsif ($arr[5] eq '-') {
$idhash{$arr[0]}{$arr[2]}{$arr[1]}{'pos'}=$arr[4];
$idhash{$arr[0]}{$arr[2]}{$arr[1]}{'str'}=$arr[5];
}
else {
die "Error: strand error at line($linenum): $line\n";
}
}
close BAMSTATSIN;
open (ONLYONE, "> $output.oneonly") || die "Error: can not write onlyonly: $output.oneonly";
my %links=();
my %read2links=();
my @allreads=();
my $unique_mapped_read_num=0;
@allreads=keys (%idhash);
foreach my $indread (@allreads) {
my @arr=();
if (exists $idhash{$indread}{'EXCLUDEDPLZ'}) {
print FILTEROUT $indread, "\tMappOneReferenceTwice\n";
delete $idhash{$indread};
next;
}
@arr=keys %{$idhash{$indread}};
my $excludethisread=0;
foreach my $indctg (@arr) {### Control read mapped to different strand and -pos is downstream of +pos
if (exists $idhash{$indread}{$indctg}{1} and exists $idhash{$indread}{$indctg}{2}) {
if ($idhash{$indread}{$indctg}{1}{'str'} eq '+' and $idhash{$indread}{$indctg}{2}{'str'} eq '-') {
unless ($idhash{$indread}{$indctg}{2}{'pos'}> $idhash{$indread}{$indctg}{1}{'pos'}) {
$excludethisread=1;
last;
}
}
elsif ($idhash{$indread}{$indctg}{1}{'str'} eq '-' and $idhash{$indread}{$indctg}{2}{'str'} eq '+') {
unless ($idhash{$indread}{$indctg}{1}{'pos'} > $idhash{$indread}{$indctg}{2}{'pos'}) {
$excludethisread=1;
last;
}
}
else {
$excludethisread=1;
last;
}
}
}
if ($excludethisread==1) {
print FILTEROUT $indread, "\tMappingProblem\n";
delete $idhash{$indread};
next;
}
if (scalar(@arr)==1) {### Control mapped to only one reference
print ONLYONE $indread, "\t";
if (exists $idhash{$indread}{$arr[0]} and exists $idhash{$indread}{$arr[0]}{2} and exists $idhash{$indread}{$arr[0]}{1}) {
my $insetsize=abs($idhash{$indread}{$arr[0]}{2}{'pos'}-$idhash{$indread}{$arr[0]}{1}{'pos'})+1;
if ($insetsize>=$min_allowed_insert and $insetsize<=$max_allowed_insert) {
push (@insertsum, $insetsize);
$insertnum++;
print ONLYONE $insetsize, "\n";
print FINALREADLIST $indread, "\n";
$unique_mapped_read_num++;
}
}
else {
print ONLYONE "NaN\n";
}
delete $idhash{$indread};
next;
}
my @arr2=();
my @arr3=();
foreach my $contig (@arr) {
push (@arr2, $contig) if (exists $idhash{$indread}{$contig}{1});
push (@arr3, $contig) if (exists $idhash{$indread}{$contig}{2});
}
if (scalar(@arr2)<1 or scalar(@arr3)<1) {### Remove Only one mate mapped
print FILTEROUT $indread, "\tOnlyOneMateMapped\n";
delete $idhash{$indread};
next;
}
my $maxmapping=max (scalar(@arr2), scalar(@arr3));
if ($maxmapping>$maxmappingtimes) {### control mapping times
print FILTEROUT $indread, "\tMappedMoreThan", $maxmappingtimes, "Times$maxmapping\n";
delete $idhash{$indread};
next;
}
foreach my $first (@arr2) {
foreach my $second (@arr3) {
next if ($first eq $second);
# next if (exists $links{$second} and exists $links{$second}{$first});
$links{$first}{$second}{$indread}++;
$read2links{$indread}{$first}{$second}++;
$links{$second}{$first}{$indread}++;
$read2links{$indread}{$second}{$first}++;
}
}
}
#print "Test: \%links\n"; print Dumper \%links; print "\n"; exit 0; ### For test ###
#print "Test: \%read2links\n"; print Dumper \%read2links; print "\n"; exit 0; ### For test ###
close ONLYONE;
print "### Summary ###\n";
print "Num: $insertnum\n";
$estimatedinsertsize=int (&CalAverage(@insertsum));
my $stdev_insertsize=int (CalStdev(@insertsum));
print "Estimated insert size average: ", $estimatedinsertsize, "\n";
print "Estimated insert size stdev : ",$stdev_insertsize , "\n";
$maxinsert=$estimatedinsertsize+$stdev_insertsize;
print "Maximum insert size : ", $maxinsert, "\n";
@insertsum=();
@allreads=();
my @firsts=();
my %scafstrandness=();
@firsts=keys %links;
#print "Test: \n"; print "Total number first strands: ", scalar(@firsts), "\n\n"; ### For test ###
foreach my $first (@firsts) {
next unless (exists $links{$first});
my @seconds=keys %{$links{$first}};
delete $links{$first} unless (scalar(@seconds)>0);
# print "SCAFFOLD1 $first\n"; ### For test ###
foreach my $second (@seconds) {
# print "\tSCAFFOLD2 $second\n"; ### For test ###
next unless (exists $links{$first}{$second});
my @readid=();
@readid=keys %{$links{$first}{$second}};
# print "SCAFFOLD1 $first SCAFFOLD2 $second Read number: ", scalar(@readid), "\n"; ### For test ###
unless (scalar(@readid)>=$numpairs) {
# print STDERR "Info: pairs not have enough evidence: SCAFFOLD1 $first SCAFFOLD2 $second\n"; ### For test ###
delete $links{$first}{$second} if (exists $links{$first} and exists $links{$first}{$second});
delete $links{$first} unless (scalar(keys %{$links{$first}})>0);
delete $links{$second}{$first} if (exists $links{$second} and exists $links{$second}{$first});
delete $links{$second} unless (scalar(keys %{$links{$second}})>0);
next;
}
# print "SCAFFOLD1 $first SCAFFOLD2 $second Read number: ", scalar(@readid), "\n"; ### For test ###
### Decide strand
%strand=('s' => 0, 'd' => 0);
$strness='NaN';
$ratio='NaN';
my %readstrand=();
foreach my $indread (@readid) {
my @firstpos=();
my @secondpos=();
unless (exists $idhash{$indread}) {
&RemoveRead($indread);
next;
}
$readproblematic=0;
&EstimateOverlap($indread, $first, $second);
if ($readproblematic==1) {
print FILTEROUT $indread, "\tProblematicMapping\n";
&RemoveRead($indread);
next;
}
my $firstmate=0;
my $secondmate=0;
if (exists $idhash{$indread}{$first}) {
if (exists $idhash{$indread}{$first}{1}) {
push (@firstpos, 1);
push (@firstpos, $idhash{$indread}{$first}{1}{'pos'});
push (@firstpos, $idhash{$indread}{$first}{1}{'str'});
$firstmate++;
}
if (exists $idhash{$indread}{$first}{2}) {
push (@secondpos, 2);
push (@secondpos, $idhash{$indread}{$first}{2}{'pos'});
push (@secondpos, $idhash{$indread}{$first}{2}{'str'});
$secondmate++;
}
}
if (exists $idhash{$indread}{$second}) {
if (exists $idhash{$indread}{$second}{1}) {
push (@secondpos, 1);
push (@secondpos, $idhash{$indread}{$second}{1}{'pos'});
push (@secondpos, $idhash{$indread}{$second}{1}{'str'});
$secondmate++;
}
if (exists $idhash{$indread}{$second}{2}) {
push (@firstpos, 2);
push (@firstpos, $idhash{$indread}{$second}{2}{'pos'});
push (@firstpos, $idhash{$indread}{$second}{2}{'str'});
$firstmate++;
}
}
# print "Read: $indread\t$first vs $second\n"; ### For test ###
# print "Test: \@firstpos: ", join ("\t", @firstpos), "\n"; ### For test ###
# print "Test: \@secondpos: ", join ("\t", @secondpos), "\n"; ### For test ###
if ($firstmate==2 or $secondmate==2) {
if ($firstmate==2 and $secondmate==2) {
if ($firstpos[2] eq $firstpos[5]) {
$strand{'s'}++; $readstrand{$indread}{'s'}++;
}
else {
$strand{'d'}++;$readstrand{$indread}{'d'}++;
}
if ($secondpos[2] eq $secondpos[5]) {
$strand{'s'}++;$readstrand{$indread}{'s'}++;
}
else {
$strand{'d'}++;$readstrand{$indread}{'d'}++;
}
}
elsif ($firstmate==2 and $secondmate<2) {
if ($firstpos[2] eq $firstpos[5]) {
$strand{'s'}++;$readstrand{$indread}{'s'}++;
}
else {
$strand{'d'}++;$readstrand{$indread}{'d'}++;
}
}
elsif ($firstmate<2 and $secondmate==2) {
if ($secondpos[2] eq $secondpos[5]) {
$strand{'s'}++;$readstrand{$indread}{'s'}++;
}
else {
$strand{'d'}++;$readstrand{$indread}{'d'}++;
}
}
else {
print "Warnings: can not understand: $first ($firstmate) vs $second ($secondmate)\tRead: $indread\n\n";
}
}
else {
print STDERR "Warnings: not mapped to any: READ: $indread SCAFFOLD1 $first SCAFFOLD2 $second\n";
next;
}
}
# print "Test: FIRST $first SECOND $second\n"; print Dumper \%strand; print "\n"; ### for test ###
###checking strand
if (($strand{'d'}+$strand{'s'})<$numpairs) {
delete $links{$first}{$second} if (exists $links{$first} and exists $links{$first}{$second});
delete $links{$first} unless (scalar(keys %{$links{$first}})>0);
delete $links{$second}{$first} if (exists $links{$second} and exists $links{$second}{$first});
delete $links{$second} unless (scalar(keys %{$links{$second}})>0);
next;
}
my $finalstrand='';
if ($strand{'d'}==0 and $strand{'s'}>=$numpairs) {
$strness='Reverse';
$ratio=$strand{'s'};
}
elsif ($strand{'s'}==0 and $strand{'d'}>=$numpairs) {
$strness='Same';
$ratio=$strand{'d'};
}
elsif ($strand{'s'}>=$strand{'d'} and $strand{'d'}>0) {
{
$strness='Reverse';
$ratio=$strand{'s'}/$strand{'d'};
}
}
elsif ($strand{'s'}<$strand{'d'} and $strand{'s'}>0) {
if ($strand{'d'}/$strand{'s'}>=$ratiothreshold) {
$strness='Same';
$ratio=$strand{'d'}/$strand{'s'};
}
}
else {
print STDERR "Warnings: unknown strand count: FIRST $first SECOND $second Same:", $strand{'s'}, " Diff", $strand{'d'}, "\n";
print Dumper $links{$first}{$second};
}
if ($ratio>=$ratiothreshold) {
if ($strand{'s'}>$strand{'d'}) {$finalstrand='s';}
if ($strand{'s'}<$strand{'d'}) {$finalstrand='d';}
}
unless ($finalstrand=~/^[sd]{1,1}$/) {
delete $links{$first}{$second} if (exists $links{$first} and exists $links{$first}{$second});
delete $links{$first} unless (scalar(keys %{$links{$first}})>0);
delete $links{$second}{$first} if (exists $links{$second} and exists $links{$second}{$first});
delete $links{$second} unless (scalar(keys %{$links{$second}})>0);
next;
}
# print "SCAFFOLD1 $first SCAFFOLD2 $second STRAND $finalstrand\n";### For test ###
foreach my $indread (keys %readstrand) {
if (exists $readstrand{$indread}{'s'} and exists $readstrand{$indread}{'d'}) {
print FILTEROUT "$indread\tControversyStrandingbySelf\n";
&RemoveRead($indread);
next;
}
unless (exists $readstrand{$indread} and exists $readstrand{$indread}{$finalstrand}) {
print FILTEROUT "$indread\tControversyStrandingWithOthers\n";
&RemoveRead($indread);
next;
}
}
unless (exists $scafstrandness{$first} and exists $scafstrandness{$first}{$second}) {
$scafstrandness{$first}{$second}=$finalstrand;
# $scafstrandness{$second}{$first}=$finalstrand;
}
else {
die "Error: repeated strand: SCAFFOLD1 $first SCAFFOLD2 $second STRAND $finalstrand\n";
}
}
}
#print "\n\n\n"; print Dumper %scafstrandness; print "\n\n\n"; exit 0;### For test ###
###Double check
@firsts=();
@firsts=keys %links;
foreach my $first (@firsts) {
next unless (exists $links{$first});
my @seconds=keys %{$links{$first}};
delete $links{$first} unless (scalar(@seconds)>0);
foreach my $second (@seconds) {
next unless (exists $links{$first}{$second});
my @readid=();
@readid=keys %{$links{$first}{$second}};
unless (scalar(@readid)>0) {
delete $links{$first}{$second};
delete $links{$first} unless (scalar(keys %{$links{$first}})>0);
delete $links{$second}{$first} if (exists $links{$second} and exists $links{$second}{$first});
delete $links{$second} if (exists $links{$second} and scalar(keys %{$links{$second}})==0);
}
my $numlinks=0;
foreach my $indread (@readid) {
if (exists $idhash{$indread}) {
$numlinks++;
}
else {
delete $links{$first}{$second}{$indread};
}
}
unless ($numlinks>=$numpairs) {
print STDERR "Info: pairs not have enough evidence2: SCAFFOLD1 $first SCAFFOLD2 $second\n";
delete $links{$first}{$second} if (exists $links{$first} and exists $links{$first}{$second});
delete $links{$first} unless (scalar(keys %{$links{$first}})>0);
delete $links{$second}{$first} if (exists $links{$second} and exists $links{$second}{$first});
delete $links{$second} if (exists $links{$second} and scalar(keys %{$links{$second}})==0);
next;
}
}
}
@firsts=();
@firsts=keys %links;
foreach my $first (@firsts) {
next unless (exists $links{$first});
my @seconds=keys %{$links{$first}};
delete $links{$first} unless (scalar(@seconds)>0);
foreach my $second (@seconds) {
next unless (exists $links{$first}{$second});
my @readid=();
@readid=keys %{$links{$first}{$second}};
unless (scalar(@readid)>0) {
delete $links{$first}{$second};
delete $links{$first} unless (scalar(keys %{$links{$first}})>0);
delete $links{$second}{$first} if (exists $links{$second} and exists $links{$second}{$first});
delete $links{$second} if (exists $links{$second} and scalar(keys %{$links{$second}})==0);
}
$testwindows=0;
unless (scalar(@readid)>=$numpairs) {
print STDERR "Info: pairs not have enough evidence\n";
next;
}
my $plusstart=0; my %finalplus=();
my $minusstart=0; my %finalminus=();
my $arrstart=0; %finalarray=();
%strand=('s' => 0, 'd' => 0);
$strness='NaN';
$ratio='NaN';
$test_overlap=0;
$printthislink=0;
%read2gap=();
my @disance=();
my @gaps=();
foreach my $indread (@readid) {
my @firstpos=();
my @secondpos=();
die "Error: Non-existed2 readname: $indread\n" unless (exists $idhash{$indread});
my $firstmate=0;
my $secondmate=0;
if (exists $idhash{$indread}{$first}) {
if (exists $idhash{$indread}{$first}{1}) {
print FILTERED "$indread\t1\t$first\t", $idhash{$indread}{$first}{1}{'pos'}, "\t", $idhash{$indread}{$first}{1}{'str'}, "\n";
push (@firstpos, 1);
push (@firstpos, $idhash{$indread}{$first}{1}{'pos'});
push (@firstpos, $idhash{$indread}{$first}{1}{'str'});
$firstmate++;
}
if (exists $idhash{$indread}{$first}{2}) {
print FILTERED "$indread\t2\t$first\t", $idhash{$indread}{$first}{2}{'pos'}, "\t", $idhash{$indread}{$first}{2}{'str'}, "\n";
push (@secondpos, 2);
push (@secondpos, $idhash{$indread}{$first}{2}{'pos'});
push (@secondpos, $idhash{$indread}{$first}{2}{'str'});
$secondmate++;
}
}
if (exists $idhash{$indread}{$second}) {
if (exists $idhash{$indread}{$second}{1}) {
print FILTERED "$indread\t1\t$second\t", $idhash{$indread}{$second}{1}{'pos'}, "\t", $idhash{$indread}{$second}{1}{'str'}, "\n";
push (@secondpos, 1);
push (@secondpos, $idhash{$indread}{$second}{1}{'pos'});
push (@secondpos, $idhash{$indread}{$second}{1}{'str'});
$secondmate++;
}
if (exists $idhash{$indread}{$second}{2}) {
print FILTERED "$indread\t2\t$second\t", $idhash{$indread}{$second}{2}{'pos'}, "\t", $idhash{$indread}{$second}{2}{'str'}, "\n";
push (@firstpos, 2);
push (@firstpos, $idhash{$indread}{$second}{2}{'pos'});
push (@firstpos, $idhash{$indread}{$second}{2}{'str'});
$firstmate++;
}
}
else {
die "Error: Non-existed2 readname:contig $indread\n";
}
# print "Read: $indread\t$first vs $second\n";
# print "Test: \@firstpos: ", join ("\t", @firstpos), "\n"; ### For test ###
# print "Test: \@secondpos: ", join ("\t", @secondpos), "\n"; ### For test ###
if ($firstmate==2 or $secondmate==2) {
if ($firstmate==2 and $secondmate==2) {
print OVERLAP "Info: defined overlap: $first vs $second\tRead: $indread\n";
$finalarray{$arrstart}{'readid'}=$indread;
@{$finalarray{$arrstart++}{'arr'}}=@firstpos;
if ($firstpos[2] eq '+') {
$finalplus{$plusstart}{'readid'}=$indread;
@{$finalplus{$plusstart++}{'arr'}}=@firstpos;
}
elsif ($firstpos[2] eq '-') {
$finalminus{$minusstart}{'readid'}=$indread;
@{$finalminus{$minusstart++}{'arr'}}=@firstpos;
}
if ($firstpos[2] eq $firstpos[5]) {
$strand{'s'}++;
}
else {
$strand{'d'}++;
}
$finalarray{$arrstart}{'readid'}=$indread;
@{$finalarray{$arrstart++}{'arr'}}=@secondpos;
if ($secondpos[2] eq '+') {
$finalplus{$plusstart}{'readid'}=$indread;
@{$finalplus{$plusstart++}{'arr'}}=@secondpos;
}
elsif ($secondpos[2] eq '-') {
$finalminus{$minusstart}{'readid'}=$indread;
@{$finalminus{$minusstart++}{'arr'}}=@secondpos;
}
if ($secondpos[2] eq $secondpos[5]) {
$strand{'s'}++;
}
else {
$strand{'d'}++;
}
}
elsif ($firstmate==2 and $secondmate<2) {
print OVERLAP "Info: possible overlap: $first vs $second\tRead: $indread\n" if ($secondmate==1);
$finalarray{$arrstart}{'readid'}=$indread;
# print join("\t", @firstpos), "\n"; ### For test ###
@{$finalarray{$arrstart++}{'arr'}}=@firstpos;
if ($firstpos[2] eq '+') {
$finalplus{$plusstart}{'readid'}=$indread;
@{$finalplus{$plusstart++}{'arr'}}=@firstpos;
}
elsif ($firstpos[2] eq '-') {
$finalminus{$minusstart}{'readid'}=$indread;
@{$finalminus{$minusstart++}{'arr'}}=@firstpos;
}
if ($firstpos[2] eq $firstpos[5]) {
$strand{'s'}++;
}
else {
$strand{'d'}++;
}
}
elsif ($firstmate<2 and $secondmate==2) {
print OVERLAP "Info: possible overlap: $first vs $second\tRead: $indread\n" if ($firstmate==1);
# print join("\t", @secondpos), "\n"; ### For test ###
$finalarray{$arrstart}{'readid'}=$indread;
@{$finalarray{$arrstart++}{'arr'}}=@secondpos;
if ($secondpos[2] eq '+') {
$finalplus{$plusstart}{'readid'}=$indread;
@{$finalplus{$plusstart++}{'arr'}}=@secondpos;
}
elsif ($secondpos[2] eq '-') {
$finalminus{$minusstart}{'readid'}=$indread;
@{$finalminus{$minusstart++}{'arr'}}=@secondpos;
}
if ($secondpos[2] eq $secondpos[5]) {
$strand{'s'}++;
}
else {
$strand{'d'}++;
}
}
else {
print "Warnings: can not understand: $first ($firstmate) vs $second ($secondmate)\tRead: $indread\n\n";
}
unless (&EstimateOverlap($indread, $first, $second)) {
die "Error: SUB(EstimateOverlap) returns error\n";
}
if (0) {### For test ###
print "\%read2gap\n";
print $indread, "\t$first: ", $seqlength{$first}, "\t$second: ", $seqlength{$second}, "\n";
print Dumper $idhash{$indread}{$first};
print Dumper $idhash{$indread}{$second};
print Dumper $read2gap{$indread};
print "\n";
}
if (exists $read2gap{$indread}and exists $read2gap{$indread}{'dist'}) {
push (@disance, $read2gap{$indread}{'dist'});
}
if (exists $read2gap{$indread}and exists $read2gap{$indread}{'gap'}) {
push (@gaps, $read2gap{$indread}{'gap'});
}
}
else {
next;
}
}
if (0) {### For test ###
print "\%read2gap\n";
print Dumper \%read2gap;
print "\n";
exit 0;
}
my $meandist=0;
my $stdevdist=0;
$meandist=int(&CalAverage(@disance));
$stdevdist=int(&CalStdev(@disance));
my $meangap=0;
my $stdevgap=0;
$meangap=int(&CalAverage(@gaps));
$stdevgap=int(&CalStdev(@gaps));
if (abs($meandist)<=$maxinsert) {# and abs($meangap)<=$maxinsert) {### Limit Mean dist and gap
if ($maxmappingtimes==1) {
if (abs($meangap)<=$maxinsert) {
$printthislink=1;
}
else {
$printthislink=0;
}
}
else {
$printthislink=1;
}
}
else {
$printthislink=0;
}
if ($strand{'d'}==0 and $strand{'s'}>=$numpairs) {
$strness='RC';
$ratio=$strand{'s'};
}
elsif ($strand{'s'}==0 and $strand{'d'}>=$numpairs) {
$strness='ST';
$ratio=$strand{'d'};
}
else {
print STDERR "Warnings: unknown strand count: Same:",$strand{'s'}," Diff",$strand{'d'},"\n";
}
# print "Test: \%finalarray\n";print Dumper \%finalarray;print "\n"; ### For test ###
###check distance
my $total_windows=0;
if (scalar (keys %finalplus)>=$numpairs) {
unless(&GetDistance($first, $second, \%finalplus)) {
print STDERR "Error: \%finalplus\n";
die "\n";
}
$total_windows=scalar (keys %finalplus)-$numpairs+1+$total_windows;
}
else {
print DISTANCE "#NNNplus\t$first\t+\t$second\n";
}
if (scalar (keys %finalminus)>=$numpairs) {
unless(&GetDistance($first, $second, \%finalminus)) {
print STDERR "Error: \%finalminus\n";
die "\n";
}
$total_windows=scalar (keys %finalminus)-$numpairs+1+$total_windows;
}
else {
print DISTANCE "#NNNminus\t$first\t-\t$second\n";
}
if ($testwindows>0 and $printthislink==1) {
print SCAFPAIR $first, "\t", $seqlength{$first}, "\t", $second, "\t", $seqlength{$second}, "\t", $strand{'d'}, '/', $strand{'s'}, "\t", $ratio, "\t$strness\t", $testwindows, "\t", $total_windows, "\t", $testwindows/$total_windows, "\t$test_overlap\t$meandist\t$stdevdist\t$meangap\t$stdevgap\n";
# print "### Test ###\n"; print Dumper $links{$first}{$second}; exit 0;### For test ###
foreach my $readidv (@readid) {
print FINALREADLIST $readidv, "\n" unless (exists $printreads{$readidv});
$printreads{$readidv}++;
}
print FINALREFSLIST $first, "\n" unless (exists $printrefs{$first});
$printrefs{$first}++;
print FINALREFSLIST $second, "\n" unless (exists $printrefs{$second});
$printrefs{$second}++;
}
}
}
close FILTEROUT;
close FILTERED;
close DISTANCE;
close OVERLAP;
close SCAFPAIR;
close FINALREFSLIST;
close FINALREADLIST;
print "Output unique reads: $unique_mapped_read_num\n";
print "Output joinable reads: ", scalar(keys %printreads), "\n";
print "Output references: ", scalar(keys %printrefs), "\n";
### Global: $numpairs
sub GetDistance {
my ($GDfirst, $GDsecond, $GDhash)=@_;
my $GDsubinfo='SUB(GetDistance)';
my ($GDtest_sort, $GDreadorder)=&SortIdHash($GDhash);
unless ($GDtest_sort) {
print STDERR $GDsubinfo, "Error: SortIdHash failed\n";
return 0;
}
for (my $GDi=0; $GDi<=(scalar(@{$GDreadorder})-$numpairs); $GDi++) {
my @GDtemparrpos1=();
my @GDtempstrand1=();
my @GDtemparrpos2=();
my @GDtempstrand2=();
my @GDtempfix=();
my @GDtempflanking=();
for (my $GDj=0; $GDj<$numpairs; $GDj++) {
my $GDorder=${$GDreadorder}[$GDi+$GDj];
# print " $GDi $GDj ", ${$GDhash}{$GDorder}{'readid'}, " $GDfirst vs $GDsecond\n";### For test ###
unless (exists ${$GDhash}{$GDorder} and exists ${$GDhash}{$GDorder}{'arr'} and defined ${$GDhash}{$GDorder}{'arr'}[1] and ${$GDhash}{$GDorder}{'arr'}[1]=~/^\d+$/ and defined ${$GDhash}{$GDorder}{'arr'}[4] and ${$GDhash}{$GDorder}{'arr'}[4]=~/^\d+$/) {
print STDERR $GDsubinfo, "Error: second pos: $GDfirst vs $GDsecond: \n";
return 0;
}
my ($GDmatenum1, $GDmatepos1, $GDmatestr1, $GDmatenum2, $GDmatepos2, $GDmatestr2)=@{${$GDhash}{$GDorder}{'arr'}};
push (@GDtemparrpos1, $GDmatepos1);
push (@GDtempstrand1, $GDmatestr1);
push (@GDtemparrpos2, $GDmatepos2);
push (@GDtempstrand2, $GDmatestr2);
my ($GDtest, $GDfixed, $GDgapsize)= &EstimateGap($GDfirst, $GDsecond, ${$GDhash}{$GDorder});
unless ($GDtest) {
print STDERR $GDsubinfo, "Error: EstimateGap failed: $GDfirst vs $GDsecond\n";
print Dumper ${$GDhash}{$GDorder};
return 0;
}
# push (@GDtempfix, $GDfixed);
push (@GDtempflanking, $GDgapsize);
# print " $GDi $GDj ", ${$GDhash}{$GDorder}{'readid'}, " $GDfirst vs $GDsecond $GDgapsize\n"; ### For test ###
}
my @GDnewtemparrpos2=sort {$a<=>$b} @GDtemparrpos2;
my $GDmaxdistance1=$GDtemparrpos1[-1]-$GDtemparrpos1[0];
my $GDmaxdistance2=$GDnewtemparrpos2[-1]-$GDnewtemparrpos2[0];
# $test_overlap=0;
# if (sum(@GDtempfix) >1) {$test_overlap=1;} ### would defined overlap if 1 of then show overlap
my $GDaveragedistancebetween=&CalAverage(@GDtempflanking);
# print DISTANCE"No/Total-NumReads\tRef1name\tRef1len\tRef1std\tRef2name\tRef2len\tRef2std\tS/D\tratio\tAssemblyStd\tDist1\tDist1Detail\tDist2\tDist2Detail\tTest_overlap\tMeaninternalSize\tDetail\n";
print DISTANCE $GDi,'/',scalar(keys %{$GDhash}),"-$numpairs\t", "$GDfirst\t", $seqlength{$GDfirst}, "\t", join ('', @GDtempstrand1),"\t", $GDsecond, "\t", $seqlength{$GDsecond}, "\t", join ('', @GDtempstrand2), "\t", $strand{'s'}, '/', $strand{'d'}, "\t$ratio\t$strness\t", "$GDmaxdistance1\t(", join (",", @GDtemparrpos1), ")\t$GDmaxdistance2\t(", join (',', @GDtemparrpos2), ")\t$test_overlap\t", $GDaveragedistancebetween, "\t(", join(',', @GDtempflanking), ")\n";
if ($windows2!=0 and $GDmaxdistance1<=$windows1 and $GDmaxdistance2<=$windows2 and $GDaveragedistancebetween<=$maxinsert) {
$testwindows++;
}
}
return 1;
}
### Global: %idhash
sub EstimateGap {
my ($EGref1, $EGref2, $EGhashref)=@_;
my $EGsubinfo='SUB(EstimateGap)';
my @EGfixedgap=();
my $EGestgapsize=0;
my $EGfinalsize=0;
my $EGfinalfix=0;
my $EGflank1=0;
my $EGflank2=0;
my $EGreadid=${$EGhashref}{'readid'};
my ($EGmatenum1, $EGmatepos1, $EGmatestr1, $EGmatenum2, $EGmatepos2, $EGmatestr2)=@{${$EGhashref}{'arr'}};
unless (defined $EGmatenum1 and $EGmatenum1=~/^[12]{1}$/ and defined $EGmatenum2 and $EGmatenum2=~/^[12]{1}$/ and ($EGmatenum1 != $EGmatenum2)) {
print STDERR $EGsubinfo, "Error: read $EGreadid array: $EGmatenum1, $EGmatepos1, $EGmatestr1, $EGmatenum2, $EGmatepos2, $EGmatestr2\n";
return 0;
}
unless (exists $idhash{$EGreadid} and exists $idhash{$EGreadid}{$EGref1}) {
print STDERR $EGsubinfo, "Error: idhash not existed for read $EGreadid\n";
return 0;
}
unless (exists $idhash{$EGreadid}{$EGref1}{$EGmatenum1}) {
print STDERR $EGsubinfo, "Error: READ $EGreadid R$EGmatenum1 REF $EGref1 ARRAY $EGmatenum1, $EGmatepos1, $EGmatestr1\n";
return 0;
}
unless (exists $idhash{$EGreadid}{$EGref2}{$EGmatenum2}) {
print STDERR $EGsubinfo, "Error: READ $EGreadid R$EGmatenum2 REF $EGref1 ARRAY $EGmatenum2, $EGmatepos2, $EGmatestr2\n";
return 0;
}
if (exists $idhash{$EGreadid}{$EGref1}{$EGmatenum2}) {
my $EGgap1=abs($idhash{$EGreadid}{$EGref1}{$EGmatenum2}{'pos'}-$EGmatepos1)+1;
push (@EGfixedgap, $EGgap1);
}
if (exists $idhash{$EGreadid}{$EGref2}{$EGmatenum1}) {
my $EGgap2=abs($idhash{$EGreadid}{$EGref2}{$EGmatenum1}{'pos'}-$EGmatepos2)+1;
push (@EGfixedgap, $EGgap2);
}
if (scalar(@EGfixedgap)==2) {
$EGfinalsize= max @EGfixedgap;
$EGfinalfix=1;
}
elsif (scalar(@EGfixedgap)==1) {
$EGfinalsize=$EGfixedgap[0];
$EGfinalfix=1;
}
else {
unless (exists $seqlength{$EGref1} and $seqlength{$EGref1}>=$EGmatepos1) {
print STDERR $EGsubinfo, "Error: unknown length: $EGref1\n";
return 0;
}
unless (exists $seqlength{$EGref2} and $seqlength{$EGref2}>=$EGmatepos2) {
print STDERR $EGsubinfo, "Error: unknown length: $EGref2\n";
return 0;
}
$EGflank1=0;
$EGflank2=0;
if ($EGmatestr1 eq '+') {
$EGflank1=$seqlength{$EGref1}-$EGmatepos1+1;
}
elsif ($EGmatestr1 eq '-') {
$EGflank1=$EGmatepos1;
}
else {
print STDERR $EGsubinfo, "Error: unknown R$EGmatenum1 strand: READID $EGreadid REF $EGref1 STRAND $EGmatestr1\n";
return 0;
}
if ($EGmatestr2 eq '+') {
$EGflank2=$seqlength{$EGref2}-$EGmatepos2+1;
}
elsif ($EGmatestr2 eq '-') {
$EGflank2=$EGmatepos2;
}
else {
print STDERR $EGsubinfo, "Error: unknown R$EGmatenum2 strand: READID $EGreadid REF $EGref2 STRAND $EGmatestr2\n";
return 0;
}
if (defined $EGflank1 and $EGflank1=~/^\d+$/ and defined $EGflank2 and $EGflank2=~/^\d+$/) {
$EGfinalsize=$EGflank1+$EGflank2;
}
else {
print STDERR $EGsubinfo, "Error: unknown R$EGmatenum1 flanking size: READID $EGreadid REF $EGref1 STRAND $EGmatestr1\n" unless (defined $EGflank1 and $EGflank1=~/^\d+$/);
print STDERR $EGsubinfo, "Error: unknown R$EGmatenum2 flanking size: READID $EGreadid REF $EGref2 STRAND $EGmatestr2\n" unless (defined $EGflank2 and $EGflank2=~/^\d+$/);
return 0;
}
}
# print "READID $EGreadid REF $EGref1 ($seqlength{$EGref1}) POS1 $EGmatepos1 STRAND $EGmatestr1 REF $EGref2($seqlength{$EGref2}) POS2 $EGmatepos2 STRAND $EGmatestr2 Fix $EGfinalfix FLANK1 $EGflank1 FLANK2 $EGflank2\n"; ### for test ###
return (1, $EGfinalfix, $EGfinalsize);
}
sub CalAverage {
return sum(@_)/@_;
}
sub CalStdev {
my $CSsum=0;
my $CSaverage=&CalAverage(@_);
foreach (@_) {
$CSsum=$CSsum+($_-$CSaverage) ** 2;
}
$CSsum = $CSsum / scalar(@_);
$CSsum= sqrt ($CSsum);
return $CSsum;
}
### Global: $numpairs
### Dependency:
### Note:
sub SortIdHash {
my $SIHhash=shift;
my @SIHarr=();
my $SIHsubinfo='SUB(SortIdHash)';
my %SIHtemp=();
if (scalar(keys %{$SIHhash})<$numpairs) {
print STDERR $SIHsubinfo, "Error: invalid input hash: ", scalar(keys %{$SIHhash}), "<5 keys\n";
return 0;
}
foreach my $SIHnum (keys %{$SIHhash}) {