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splice_score_novel.pl
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#!/usr/bin/perl
use strict;
use warnings;
(my $usage = <<OUT) =~ s/\t+//g;
perl splice_score_novel.pl dir_s dir_in info f_ref f_bed
OUT
die $usage unless @ARGV == 5;
my ($dir_s,$dir_in, $info,$f_ref,$f_bed) = @ARGV;
print "Reading genome.......";
my $genome = $f_ref;
#print $genome,"\n";
#my ($dir_in) = @ARGV;
open(REF,"$genome");
my %gen;
my $id;
while(<REF>)
{
chomp;
my $first = substr($_,0,1);
if($first eq ">")
{
my @line = split(/\s+/, substr($_,1));
$id=$line[0];
## remove chr if it has ##
$id=~s/chr//g;
}else
{
$gen{$id}.=uc($_);
}
}
print "Done!\n";
print "Reading annotation...";
my $bed = $f_bed;
my %strand;
open(IN, "$bed");
while(<IN>)
{
chomp;
next if $_ =~m/^#/;
my @l = split(/\t/,);
# next if $l[2] ne "gene";
my @m = split(/\:/, $l[3]);
# for(my $i=0;$i<scalar @m;$i++)
# {
# if($m[$i]=~/gene_name/)
# {
# my @m2=split(/\"/,$m[$i]);
$strand{uc($m[0])} = $m[2];
# }
# }
}
print "Done!\n";
my $DIR = "$dir_s/CB_splice/";
##### calculate 5’ splice strength #####
my $tmpfile1=$DIR."me2x5";
my %me2x5 = &makescorematrix($tmpfile1);
my $tmpfile2=$DIR."splicemodels/splice5sequences";
my %seq = &makesequencematrix($tmpfile2);
my %bgd;
$bgd{'A'} = 0.27;
$bgd{'C'} = 0.23;
$bgd{'G'} = 0.23;
$bgd{'T'} = 0.27;
sub makesequencematrix{
my $file = shift;
my %matrix;my $n=0;
open(SCOREF, $file) || die "Can't open $file!\n";
while(<SCOREF>) {
chomp;
$_=~ s/\s//;
$matrix{$_} = $n;
$n++;
}
close(SCOREF);
return %matrix;
}
sub makescorematrix{
my $file = shift;
my %matrix;my $n=0;
open(SCOREF, $file) || die "Can't open $file!\n";
while(<SCOREF>) {
chomp;
$_=~ s/\s//;
$matrix{$n} = $_;
$n++;
}
close(SCOREF);
return %matrix;
}
sub getrest5{
my $seq = shift;
my @seqa = split(//,uc($seq));
return $seqa[0].$seqa[1].$seqa[2].$seqa[5].$seqa[6].$seqa[7].$seqa[8];
}
sub scoreconsensus5{
my $seq = shift;
my @seqa = split(//,uc($seq));
my %bgd;
$bgd{'A'} = 0.27; $bgd{'C'} = 0.23; $bgd{'G'} = 0.23; $bgd{'T'} = 0.27;
my %cons1;
$cons1{'A'} = 0.004; $cons1{'C'} = 0.0032; $cons1{'G'} = 0.9896; $cons1{'T'} = 0.0032;
my %cons2;
$cons2{'A'} = 0.0034; $cons2{'C'} = 0.0039; $cons2{'G'} = 0.0042; $cons2{'T'} = 0.9884;
my $addscore = $cons1{$seqa[3]}*$cons2{$seqa[4]}/($bgd{$seqa[3]}*$bgd{$seqa[4]});
return $addscore;
}
sub log2{
my ($val) = @_;
return log($val)/log(2);
}
##### calculate 5’ splice strength #####
##### calculate 3’ splice strength #####
sub hashseq{
#returns hash of sequence in base 4
# &hashseq('CAGAAGT') returns 4619
my $seq = shift;
$seq = uc($seq);
$seq =~ tr/ACGT/0123/;
my @seqa = split(//,$seq);
my $sum = 0;
my $len = length($seq);
my @four = (1,4,16,64,256,1024,4096,16384);
my $i=0;
while ($i<$len) {
$sum+= $seqa[$i] * $four[$len - $i -1] ;
$i++;
}
return $sum;
}
my @metables = &makemaxentscores;
sub makemaxentscores{
my $dir = $DIR."splicemodels/";
my @list = ('me2x3acc1','me2x3acc2','me2x3acc3','me2x3acc4','me2x3acc5','me2x3acc6','me2x3acc7','me2x3acc8','me2x3acc9');
my @metables;
my $num = 0 ;
foreach my $file (@list) {
my $n = 0;
open (SCOREF,"<".$dir.$file) || die "Can't open $file!\n";
while(<SCOREF>) {
chomp;
$_=~ s/\s//;
$metables[$num]{$n} = $_;
$n++;
}
close(SCOREF);
#print STDERR $file."\t".$num."\t".$n."\n";
$num++;
}
return @metables;
}
sub makewmmscores{
my $dir = $DIR."splicemodels/";
my @list = ('me1s0acc1','me1s0acc2','me1s0acc3','me1s0acc4','me1s0acc5','me1s0acc6','me1s0acc7','me1s0acc8','me1s0acc9');
my @metables;
my $num = 0 ;
foreach my $file (@list) {
my $n = 0;
open (SCOREF,"<".$dir.$file) || die "Can't open $file!\n";
while(<SCOREF>) {
chomp;
$_=~ s/\s//;
$metables[$num]{$n} = $_;
$n++;
}
close(SCOREF);
#print STDERR $file."\t".$num."\t".$n."\n";
$num++;
}
return @metables;
}
sub makemmscores{
my $dir = $DIR."splicemodels/";
my @list = ('me2s0acc1','me2s0acc2','me2s0acc3','me2s0acc4','me2s0acc5','me2s0acc6','me2s0acc7','me2s0acc8','me2s0acc9');
my @metables;
my $num = 0 ;
foreach my $file (@list) {
my $n = 0;
open (SCOREF,"<".$dir.$file) || die "Can't open $file!\n";
while(<SCOREF>) {
chomp;
$_=~ s/\s//;
$metables[$num]{$n} = $_;
$n++;
}
close(SCOREF);
#print STDERR $file."\t".$num."\t".$n."\n";
$num++;
}
return @metables;
}
sub maxentscore{
my $seq = shift;
my $table_ref = shift;
my @metables = @$table_ref;
my @sc;
$sc[0] = $metables[0]{&hashseq(substr($seq,0,7))};
$sc[1] = $metables[1]{&hashseq(substr($seq,7,7))};
$sc[2] = $metables[2]{&hashseq(substr($seq,14,7))};
$sc[3] = $metables[3]{&hashseq(substr($seq,4,7))};
$sc[4] = $metables[4]{&hashseq(substr($seq,11,7))};
$sc[5] = $metables[5]{&hashseq(substr($seq,4,3))};
$sc[6] = $metables[6]{&hashseq(substr($seq,7,4))};
$sc[7] = $metables[7]{&hashseq(substr($seq,11,3))};
$sc[8] = $metables[8]{&hashseq(substr($seq,14,4))};
my $finalscore = $sc[0] * $sc[1] * $sc[2] * $sc[3] * $sc[4] / ($sc[5] * $sc[6] * $sc[7] * $sc[8]);
return $finalscore;
}
sub getrest3{
my $seq = shift;
my $seq_noconsensus = substr($seq,0,18).substr($seq,20,3);
return $seq_noconsensus;
}
sub scoreconsensus3{
my $seq = shift;
my @seqa = split(//,uc($seq));
my %bgd;
$bgd{'A'} = 0.27; $bgd{'C'} = 0.23; $bgd{'G'} = 0.23; $bgd{'T'} = 0.27;
my %cons1;
$cons1{'A'} = 0.9903; $cons1{'C'} = 0.0032; $cons1{'G'} = 0.0034; $cons1{'T'} = 0.0030;
my %cons2;
$cons2{'A'} = 0.0027; $cons2{'C'} = 0.0037; $cons2{'G'} = 0.9905; $cons2{'T'} = 0.0030;
my $addscore = $cons1{$seqa[18]} * $cons2{$seqa[19]}/ ($bgd{$seqa[18]} * $bgd{$seqa[19]});
return $addscore;
}
##### calculate 3’ splice strength #####
sub revcom
{
my $in = shift;
$in=~tr/[AGCT]/[TCGA]/;
return reverse($in);
}
open(OUT, ">$dir_in/novel.splice.scores");
print OUT "Sample\tChr\tPos\tRef_snp\tAlt_snp\tStrand\tJunc\tRef_seq\tAlt_seq\tRef_score\tAlt_score\n";
my $indel_skipped = 0;
print "Processing intputs.......";
my $dir = $dir_in;
#foreach my $input (glob("$dir/spliceinator.splice.score.*.v2.filtered.5"))
foreach my $input (glob("$dir/$info"))
{
my %hash;
open(IN, "$input");
while(<IN>)
{
chomp;
my @line = split(/\t/,);
my $id = join("\t", $line[4], $line[5]);
next if $line[0] eq "Unknown";
$hash{$id} = $strand{uc($line[0])};
}
open(DATA, "$input.detailed.alignment.5.max");
#print "while","\n";
#print $input,"\n";
while(<DATA>)
{
#my $line=$_;
#chomp($line);
chomp;
my @templ=split(/\t/,);
#print $line,"\n";
my $nl=scalar @templ;
#print $nl,"\n";
#<STDIN>;
if($nl==5)
{
my @l = split(/\t/,);
#### using length 20 as a cut-off ####
#print $l[3],"\t",$l[4],"\n";
if(length($l[3])>=20 || length($l[4])>=20) { next; }
my $nxt = <DATA>;
my @m = split(/\t/, $nxt);
my @number=($m[5]=~m/(\d+)\w/g);
### l2 mutation position, 21M6034N54M, number[0]-> 21; number[1]->6034
my $d1 = abs($m[3] + $number[0]- $l[2]);
my $d2 = abs($m[3] + $number[0] + $number[1] - $l[2]);
my $jd = join("\t", $l[1], $l[2]);
next if !exists $hash{$jd};
my ($type, $start, $ref, $mut, $junc);
$l[3]=~s/-//;
$l[4]=~s/-//;
my $dif=length($l[3])-length($l[4]);
if($hash{$jd} eq "+")
{
if($d1 > $d2)
{
$type = "3ss";
$start = $m[3] + $number[0] + $number[1] -21;
$junc = $m[3] + $number[0] + $number[1] -1;
$ref = substr($gen{$l[1]}, $start, 23);
if($l[2]>$start && $l[2]<=$start+23)
{
if($l[2]<$m[3] + $number[0] + $number[1]) #intron
{
if($dif==0)
{#tested
$mut = substr($gen{$l[1]}, $start-$dif, $l[2]-$start-1+$dif).$l[4].substr($gen{$l[1]}, $l[2]+length($l[4])-1, $start+24-$l[2]-length($l[4]));
}elsif($dif>0)
{
if($start+24-$l[2]-$dif<=0)
{
$mut="AAAAAAAAAAAAAAAAAAAAAAA";
}else
{
$mut = substr($gen{$l[1]}, $start-$dif, $l[2]-$start-1+$dif).$l[4].substr($gen{$l[1]}, $l[2]+$dif-1, $start+24-$l[2]-$dif);
}
}else
{#tested
$mut = substr($gen{$l[1]}, $start, $l[2]-$start).$l[4].substr($gen{$l[1]}, $l[2], $start+23-$l[2]);
$mut = substr($mut, length($mut)-23);
}
}else
{#rare
if($dif==0)
{
$mut = substr($gen{$l[1]}, $start, $l[2]-$start-1).$l[4].substr($gen{$l[1]}, $l[2]+length($l[4])-1, $start+24-$l[2]-length($l[4]));
}elsif($dif>0)
{
$mut = substr($gen{$l[1]}, $start, $l[2]-$start-1).$l[4].substr($gen{$l[1]}, $l[2]+$dif-1, $start+24-$l[2]);
}else
{
$mut = substr($gen{$l[1]}, $start, $l[2]-$start).$l[4].substr($gen{$l[1]}, $l[2], $start+23-$l[2]);
$mut = substr($mut, 0, 23);
}
}
}else
{
$mut = $ref;
}
}else
{
$type = "5ss";
$start = $m[3] + $number[0] - 4;
$junc = $m[3] + $number[0];
$ref = substr($gen{$l[1]}, $start, 9);
if($l[2]>$start && $l[2]<=$start+9)
{
if($l[2]<$m[3]+$number[0])
{#rare
if($dif==0)
{
$mut = substr($gen{$l[1]}, $start, $l[2]-$start-1).$l[4].substr($gen{$l[1]}, $l[2]+length($l[4])-1, $start+10-$l[2]-length($l[4]));
}elsif($dif>0)
{
$mut = substr($gen{$l[1]}, $start-$dif, $l[2]-$start-1+$dif).$l[4].substr($gen{$l[1]}, $l[2]+$dif-1, $start+10-$l[2]);
}else
{
$mut = substr($gen{$l[1]}, $start, $l[2]-$start).$l[4].substr($gen{$l[1]}, $l[2], $start+9-$l[2]);
$mut = substr($mut, length($mut)-9);
}
}else
{
if($dif==0)
{#tested
$mut = substr($gen{$l[1]}, $start, $l[2]-$start-1).$l[4].substr($gen{$l[1]}, $l[2]+length($l[4])-1, $start+10-$l[2]-length($l[4]));
}elsif($dif>0)
{#tested
$mut = substr($gen{$l[1]}, $start, $l[2]-$start-1).$l[4].substr($gen{$l[1]}, $l[2]+$dif-1, $start+10-$l[2]);
}else
{
$mut = substr($gen{$l[1]}, $start, $l[2]-$start).$l[4].substr($gen{$l[1]}, $l[2], $start+9-$l[2]);
$mut = substr($mut, 0, 9);
}
}
}else
{
$mut = $ref;
}
}
}else
{
if($d1 > $d2)
{
$type = "5ss";
$junc = $m[3] + $number[0] + $number[1] -1;
$start = $m[3] + $number[0] + $number[1] - 7;
$ref = substr($gen{$l[1]}, $start, 9);
if($l[2]+length($l[3])-1>$start && $l[2]<=$start+9)
{
if($l[2]<$m[3] + $number[0] + $number[1])
{
if($dif==0)
{
$mut = substr($gen{$l[1]}, $start, $l[2]-$start-1).$l[4].substr($gen{$l[1]}, $l[2]+length($l[4])-1, $start+10-$l[2]-length($l[4]));
}elsif($dif>0)
{#tested
$mut = substr($gen{$l[1]}, $start-$dif, $l[2]-$start-1+$dif).$l[4].substr($gen{$l[1]}, $l[2]+$dif-1, $start+10-$l[2]-$dif);
}else
{
$mut = substr($gen{$l[1]}, $start, $l[2]-$start).$l[4].substr($gen{$l[1]}, $l[2], $start+9-$l[2]);
$mut = substr($mut, length($mut)-9);
}
}else
{
if($dif==0)
{
$mut = substr($gen{$l[1]}, $start, $l[2]-$start-1).$l[4].substr($gen{$l[1]}, $l[2]+length($l[4])-1, $start+10-$l[2]-length($l[4]));
}elsif($dif>0)
{
$mut = substr($gen{$l[1]}, $start, $l[2]-$start-1).$l[4].substr($gen{$l[1]}, $l[2]+$dif-1, $start+10-$l[2]);
}else
{
$mut = substr($gen{$l[1]}, $start, $l[2]-$start).$l[4].substr($gen{$l[1]}, $l[2], $start+9-$l[2]);
$mut = substr($mut, 0, 9);
}
}
}else
{
$mut = $ref;
}
}else
{
$type = "3ss";
$junc = $m[3] + $number[0];
$start = $m[3] + $number[0] - 4;
$ref = substr($gen{$l[1]}, $start, 23);
if($l[2]>$start && $l[2]<=$start+23)
{
if($l[2]<$m[3]+$number[0])
{
if($dif==0)
{
$mut = substr($gen{$l[1]}, $start-$dif, $l[2]-$start-1+$dif).$l[4].substr($gen{$l[1]}, $l[2]+length($l[4])-1, $start+24-$l[2]-length($l[4]));
}elsif($dif>0)
{
$mut = substr($gen{$l[1]}, $start-$dif, $l[2]-$start-1+$dif).$l[4].substr($gen{$l[1]}, $l[2]+$dif-1, $start+24-$l[2]-$dif);
}else
{#tested
$mut = substr($gen{$l[1]}, $start, $l[2]-$start).$l[4].substr($gen{$l[1]}, $l[2], $start+23-$l[2]);
$mut = substr($mut, length($mut)-23);
}
}else
{
if($dif==0)
{
$mut = substr($gen{$l[1]}, $start, $l[2]-$start-1).$l[4].substr($gen{$l[1]}, $l[2]+length($l[4])-1, $start+24-$l[2]+$dif-length($l[4]));
}elsif($dif>0)
{
$mut = substr($gen{$l[1]}, $start, $l[2]-$start-1).$l[4].substr($gen{$l[1]}, $l[2]+$dif-1, $start+24-$l[2]);
}else
{
$mut = substr($gen{$l[1]}, $start, $l[2]-$start).$l[4].substr($gen{$l[1]}, $l[2], $start+23-$l[2]);
$mut = substr($mut, 0, 23);
}
}
}else
{
$mut = $ref;
}
}
$mut = revcom($mut);
$ref = revcom($ref);
}
my ($mut_score, $ref_score);
if($type eq "5ss")
{
$ref_score = sprintf("%.2f",&log2(&scoreconsensus5($ref)*$me2x5{$seq{&getrest5($ref)}}));
$mut_score = sprintf("%.2f",&log2(&scoreconsensus5($mut)*$me2x5{$seq{&getrest5($mut)}}));
}else
{
$ref_score = sprintf("%.2f", &log2(&scoreconsensus3($ref)*&maxentscore(&getrest3($ref),\@metables)));
$mut_score = sprintf("%.2f", &log2(&scoreconsensus3($mut)*&maxentscore(&getrest3($mut),\@metables)));
}
print OUT "$_\t$type\t$hash{$jd}\t$junc\t$ref\t$mut\t$ref_score\t$mut_score\n";
}
}
}
print "Done!\n";