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Copy pathfasta_seq_for_indel_using_refseq_bed.pl
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fasta_seq_for_indel_using_refseq_bed.pl
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#!/usr/bin/perl
## generate mutant and wild-type protein sequence fir indel ##
## 12-01-2016 ##
use strict;
(my $usage = <<OUT) =~ s/\t+//g;
This script will get the protein sequence for both wide-type and mutants
OUT
die $usage unless @ARGV == 3;
my $error=0;
#my $filename="";
#my $filename_vcf_germline="";
#my $filename_vcf_somatic="";
#my $dir=".";
my($filename,$dir,$filename_vcf_somatic)=@ARGV;
#if ($ARGV[0]=~/\w/) { $filename=$ARGV[0];} else { $error=1; }
#if ($ARGV[1]=~/\w/) { $dir=$ARGV[1];} else { $dir="."; }
#if ($ARGV[2]=~/\w/) { $filename_vcf_somatic=$ARGV[2];} else { $filename_vcf_somatic=""; }
my @dir_vcf=split(/\//,$filename_vcf_somatic);
#if ($ARGV[3]=~/\w/) { $filename_vcf_somatic=$ARGV[2];} else { $filename_vcf_somatic=""; }
my %mapping = ("TTT"=>"F","TTC"=>"F","TTA"=>"L","TTG"=>"L",
"CTT"=>"L","CTC"=>"L","CTA"=>"L","CTG"=>"L",
"ATT"=>"I","ATC"=>"I","ATA"=>"I","ATG"=>"M",
"GTT"=>"V","GTC"=>"V","GTA"=>"V","GTG"=>"V",
"TCT"=>"S","TCC"=>"S","TCA"=>"S","TCG"=>"S",
"CCT"=>"P","CCC"=>"P","CCA"=>"P","CCG"=>"P",
"ACT"=>"T","ACC"=>"T","ACA"=>"T","ACG"=>"T",
"GCT"=>"A","GCC"=>"A","GCA"=>"A","GCG"=>"A",
"TAT"=>"Y","TAC"=>"Y","TAA"=>"*","TAG"=>"*",
"CAT"=>"H","CAC"=>"H","CAA"=>"Q","CAG"=>"Q",
"AAT"=>"N","AAC"=>"N","AAA"=>"K","AAG"=>"K",
"GAT"=>"D","GAC"=>"D","GAA"=>"E","GAG"=>"E",
"TGT"=>"C","TGC"=>"C","TGA"=>"*","TGG"=>"W",
"CGT"=>"R","CGC"=>"R","CGA"=>"R","CGG"=>"R",
"AGT"=>"S","AGC"=>"S","AGA"=>"R","AGG"=>"R",
"GGT"=>"G","GGC"=>"G","GGA"=>"G","GGG"=>"G");
if ($error==0)
{
my $filename_=$filename;
#$filename_="pr;
open (OUT,">$filename_vcf_somatic.fa-indel-wt.fasta");
open (OUT_MOD,">$filename_vcf_somatic.fa-indel-mut.fasta");
#open (OUT_FS,">$filename-frame-shift.fasta");
open (LOG,">$filename_vcf_somatic.fa-indel-wt.log");
open (LOG_MOD,">$filename_vcf_somatic.fa-indel-mut.log");
open (STAT,">$filename_vcf_somatic.fa-indel.stat");
my $proteins_count=0;
my $proteins_modified=0;
my $count_stop_removed=0;
my $count_stop_introduced=0;
my $count_stop_removed_somatic=0;
my $count_stop_introduced_somatic=0;
my $protein_modifications_count=0;
my @protein_modifications_distr=();
my $protein_modifications_count_somatic=0;
my @protein_modifications_distr_somatic=();
my $count_variant_in_exon=0;
my $count_variant_in_exon_somatic=0;
my $count_variant_in_exon_old_error=0;
my $count_variant_in_exon_old_error_somatic=0;
my $count_variant_in_exon_nonsyn=0;
my $count_variant_in_exon_nonsyn_somatic=0;
my $line="";
my %chr=();
my %bed=();
my %seq=();
if (open (IN,"$filename"))
{
while ($line=<IN>)
{
chomp($line);
if ($line=~/^([^\t]*)\t([^\t]*)\t([^\t]*)\t([^\t]*)\t([^\t]*)\t([^\t]*)\t([^\t]*)\t([^\t]*)\t([^\t]*)\t([^\t]*)\t([^\t]*)\t([^\t]*)$/)
{
my $chr=$1;
my $name=$4;
$bed{$name}=$line;
$chr{$chr}.="#$name#";
} else { print LOG qq!Error parsing: $line!; }
}
close(IN);
}
my %descriptions=();
#if (open (IN,"$filename_-descriptions.txt"))
#{
# while ($line=<IN>)
# {
# chomp($line);
# if ($line=~/^([^\t]+)\t([^\t]+)/)
# {
# $descriptions{$1}=$2;
# }
# }
# close(IN);
#}
my %vcf_old=();
my %vcf_new=();
my %vcf_type=();
my %vcf_anno=();
my %vcf_gene=();
my $germline_vcf=0;
my $germline_only=0;
my $somatic_only=0;
my $both_vcf=0;
my $both_vcf_differ=0;
#print "open $filename_vcf_somatic\n"; <STDIN>;
if(!-e $filename_vcf_somatic)
{
print STAT "no vcf file\n";
last;
}
open (IN,"<$filename_vcf_somatic");
while ($line=<IN>)
{
chomp($line);
print $line,"\n";
my @temp=split("\t",$line);
my $chr="chr".$temp[0];
my $pos=$temp[1];
my $old=$temp[2];
my $new=$temp[3];
my $gene=$temp[4];
my $anno=$temp[5];
my $type=$temp[6];
$pos--;
$new=~s/\,.*$//; #####
print "pos=$pos","\t","old=$old", "\t", "new=$new","\t","anno=$anno","\t","type=$type","\n";
#<STDIN>;
#if ($vcf_old{"$chr#$pos"}=~/\w/)
#{
# if ($vcf_new{"$chr#$pos"}!~/^$new$/)
# {
#print LOG_MOD qq!$chr $pos: germline:$vcf_old{"$chr#$pos"}->$vcf_new{"$chr#$pos"} somatic:$old->$new\n!;
$vcf_old{"$chr#$pos"}=$old;
$vcf_new{"$chr#$pos"}=$new;
if($type eq "SOMATIC")
{ $vcf_type{"$chr#$pos"}="S";}
else { $vcf_type{"$chr#$pos"}="G"; }
$vcf_anno{"$chr#$pos"}=$anno;
#print OUT_ONLY qq!$line\n!;
$somatic_only++;
$vcf_gene{"$chr#$pos"}=$gene;
}
close(IN);
#close(OUT_ONLY);
foreach my $chr (sort keys %chr)
{
#$chr="chr9";
print qq!$chr\n!;
my $chr2=$chr;
$chr2=~s/chr//g;
my $f_in=$dir."/Homo_sapiens.GRCh38.dna.chromosome.".$chr2.".fa";
if (open (IN,"$f_in"))
# if (open (IN,"$dir/$chr.fa"))
{
print qq!opened $chr\n!;
my $sequence="";
$line=<IN>;
chomp($line);
my $chr_=$chr;
$chr_=~s/^chr//i;
if ($line=~/^>$chr\s*/ or $line=~/^>$chr_\s*/)
{
while ($line=<IN>)
{
chomp($line);
if ($line=~/^>/)
{
print LOG qq!Error: > not expected: $line\n!;
}
else
{
$line=~s/\s+//g;
if ($line!~/^[atcgATCGnN]+$/)
{
print LOG qq!Error: unexpected character: $line\n!;
}
else
{
$sequence .= "\U$line";
}
}
}
my $temp=$chr{$chr};
my %seq_chr_pos;
while ($temp=~s/^#([^#]+)#//)
{
my $name=$1;
my $modified=0;
#print LOG qq!\n$name: $bed{$name}\n!;
#print qq!\n$name: $bed{$name}\n!;
if ($bed{$name}=~/^([^\t]*)\t([^\t]*)\t([^\t]*)\t([^\t]*)\t([^\t]*)\t([^\t]*)\t([^\t]*)\t([^\t]*)\t([^\t]*)\t([^\t]*)\t([^\t]*)\t([^\t]*)$/)
{
my $start=$2;
my $end=$3;
my $strand=$6;
my $num=$10;
my $segment_lengths="$11,";
my $segment_starts="$12,";
my $segment_lengths_extended="$11,";
my $segment_starts_extended="$12,";
#if ($strand=~/\+/) { if ($segment_lengths_extended=~s/([0-9]+)\,$//) { my $temp=$1; $temp+=100; $segment_lengths_extended.="$temp,"; } }
#else
#{
# if ($segment_lengths_extended=~s/^([0-9]+)\,//) { my $temp=$1; $temp+=100; $segment_lengths_extended="$temp,$segment_lengths_extended,"; }
# if ($segment_starts_extended=~s/^([0-9]+)\,//) { my $temp=$1; $temp-=100; $segment_starts_extended="$temp,$segment_starts_extended,"; }
#}
#print LOG qq!$segment_lengths $segment_lengths_extended $segment_starts $segment_starts_extended\n!;
my $seq_original="";
my $segment_starts_=$segment_starts;
my $segment_lengths_=$segment_lengths;
my $accu_c=0;
while ($segment_starts_=~s/^([0-9\-]+)\,//)
{
my $segment_start=$1;
if ($segment_lengths_=~s/^([0-9]+)\,//)
{
my $segment_length=$1;
my $seq_=substr $sequence,$start+$segment_start,$segment_length;
for(my $pos=1;$pos<=$segment_length;$pos++) { $seq_chr_pos{$accu_c}=$start+$segment_start+$pos-1; $accu_c++;}
$seq_original.=$seq_;
#print LOG qq!$start+$segment_start,$segment_length: $seq_\n!;
} else { print LOG qq!Error parsing $bed{$name}\n!; }
}
my $seq=$seq_original;
my $seq_original_rc= reverse $seq_original;
$seq_original_rc=~tr/ATCG/TAGC/;
my $description_="";
my %variants=();
my %variants_=();
my %variants2=();
my %variants2_=();
my $variant_loc="";
my $seqment_lengths_sum=0;
my $seqment_count=0;
my $num_indel=0;
my $segment_start_first=0;
#my $find_indel;
$segment_start_first=0;
#$segment_starts_=$segment_starts_extended;
#$segment_lengths_=$segment_lengths_extended;
#make modification for the reference genome based on the indel and snps
for(my $i=length($seq_original)-1;$i>=0;$i--)
{
my $var_pos=$seq_chr_pos{$i};
#print $chr,"\t",$var_pos,"\n";
if(defined $vcf_old{"$chr#$var_pos"}) {
#print "finding indel\n";
print $name,"\n";
print $var_pos,"\n";
print $vcf_old{"$chr#$var_pos"},"\n";
print $vcf_new{"$chr#$var_pos"},"\n";
#<STDIN>;
#print substr($seq_original,$i-1,3),"->",$vcf_old{"$chr#$var_pos"},"->",$vcf_new{"$chr#$var_pos"},"\n";
#<STDIN>;
my $leno;
my $lenn;
if($vcf_old{"$chr#$var_pos"} eq "-") { $leno=0; }
else { $leno=length($vcf_old{"$chr#$var_pos"}); }
if($vcf_new{"$chr#$var_pos"} eq "-") { $lenn=0; }
else { $lenn=length($vcf_new{"$chr#$var_pos"}); }
$num_indel++;
my $inframe;
if(abs($leno-$lenn)%3==0) { $inframe=1; }
else { $inframe=0; }
if($strand=~/\-/) {
if($vcf_new{"$chr#$var_pos"} eq "-")
{
my $rev_pos=length($seq_original)-$i-$leno;
my $left_3=($rev_pos+1)%3;
my $int_3s=int (($rev_pos+1)/3)+1;
my $int_3e=int (($rev_pos+$leno+1)/3)+1;
if($inframe==1) { $description_.=$chr."-".$var_pos;}
else { $description_.=$chr."-".$var_pos; }
}
else {
my $rev_pos=length($seq_original)-$i-$leno;
my $left_3=($rev_pos+1)%3;
my $int_3s=int (($rev_pos+1)/3)+1;
my $int_3e=int (($rev_pos+$lenn+1)/3)+1;
if($inframe==1) { $description_.=$chr."-".$var_pos; }
else { $description_.=$chr."-".$var_pos; }
}
}
else {
if ($vcf_new{"$chr#$var_pos"} eq "-")
{
my $left_3=($i+1)%3;
#print "i=$i\n";
#print substr($seq_original,$i,40),"\n";
my $int_3s=int (($i+1)/3)+1;
#print "int_3s=$int_3s\n";
#<STDIN>;
my $int_3e=int (($i+$leno+1)/3)+1;
if($inframe==1) { $description_.=$chr."-".$var_pos;}
else { $description_.=$chr."-".$var_pos; }
}
else {
my $left_3=($i+1)%3;
my $int_3s=int (($i+1)/3)+1;
my $int_3e=int (($i+$lenn+1)/3)+1;
if($inframe==1) { $description_.=$chr."-".$var_pos;}
else { $description_.=$chr."-".$var_pos; }
}
}
#print $description_,"\n";
#if($leno != $lenn) { $num_fs++; }
my $seql=substr($seq,0,$i);
print $seql,"\n";
my $seqr=substr($seq,$i+$leno,length($seq)-length($seql)-$leno);
if($lenn>=1)
{
$seq=$seql.$vcf_new{"$chr#$var_pos"}.$seqr;
}
else { $seq=$seql.$seqr; }
}
}
my $name_=$name; $name_=~s/\-[^\-]+$//;
#print "orignal sequence\n";
#print $seq_original,"\n";
#print "modified sequence\n";
#print $seq,"\n";
#my $gene=$name; $gene=~s/^[^\-]+\-//;
my $protein="";
my $protein_original="";
my $lenb_added=length($seq_original);
if($strand=~/\-/) {
my $seq_added=substr($sequence,$start-10000,10000);
$seq_original=$seq_added.$seq_original;
}
else {
my $seq_added=substr($sequence,$end,10000);
$seq_original=$seq_original.$seq_added;
}
if($strand=~/\-/) {
my $seq_added=substr($sequence,$start-10000,10000);
$seq=$seq_added.$seq;
}
else {
my $seq_added=substr($sequence,$end,10000);
$seq=$seq.$seq_added;
}
if ($strand=~/\-/)
{
my $seq_ = reverse $seq;
$seq=$seq_;
$seq=~tr/ATCG/TAGC/;
$seq_ = reverse $seq_original;
$seq_original=$seq_;
$seq_original=~tr/ATCG/TAGC/;
}
my $stop_found=0;
for(my $n=0;$n<length($seq_original) and $stop_found==0;$n=$n+3)
{
my $triplet = substr($seq_original, $n, 3);
if (length($triplet)==3)
{
if ($mapping{$triplet}=~/\*/)
{
$stop_found=1;
if($n+3>$lenb_added+1)
{
print LOG qq!Error: Stop codon not found within the bed file:$name \n$protein_original\n!;
$protein_original="";
}
}
if ($mapping{$triplet}!~/[\w\*]/) { $mapping{$triplet}="X"; }
$protein_original.=$triplet;
}
}
#for(my $n=0;$n<length($seq_original);$n=$n+3)
#{
# my $triplet = substr($seq_original, $n, 3);
# if (length($triplet)==3)
# {
# if ($mapping{$triplet}!~/[\w\*]/) { $mapping{$triplet}="X"; }
# $protein_original.=$triplet;
# }
#}
#$modified=0;
#my $modified_somatic=0;
$stop_found=0;
my $triplet_count=0;
#my $frame_shift=0;
#if ($description_=~/\w/) { $description_.="; "; }
for(my $n=0;$n<length($seq) and $stop_found==0;$n=$n+3)
{
my $n_=$n+2;
#my $triplet_count_=$triplet_count+1;
my $triplet = substr($seq, $n, 3);
if (length($triplet)==3)
{
if ($mapping{$triplet}!~/[\w\*]/) { $mapping{$triplet}="X"; }
#print LOG_MOD qq!$name $triplet_type $n-$n_: $triplet_old->$triplet - $triplet_count_:$mapping{$triplet_old}->$mapping{$triplet}\n!;
if ($mapping{$triplet}=~/\*/)
{
$stop_found=1;
}
$protein.=$triplet;
}
$triplet_count++;
}
#$proteins_count++;
#$protein_modifications_count+=$modified;
#$protein_modifications_distr[$modified]++;
#$protein_modifications_count_somatic+=$modified_somatic;
#$protein_modifications_distr_somatic[$modified_somatic]++;
#$protein_original=~s/\*$//;
#print "protein original\n";
#print $protein_original,"\n";
#<STDIN>;
if ($protein_original=~/^([^\*]+)\*.*$/)
{
print LOG qq!Error: No Stop codon found in middle of sequence:$name \n$protein_original\n!;
$protein_original="";
}
#if (length($protein_original)>6 and $protein_original!~/^\*/)
# {
# print OUT qq!>$name (MAP:$chr:$start$strand $segment_lengths $segment_starts)\n$protein_original\n!;
# }
#$protein=~s/\*$//;
#$protein=~s/^([^\*]+)\*.*$//;
if(($protein ne $protein_original) && ($num_indel==1))
{
#$proteins_modified++;
# print "original protein\n";
# print $protein_original,"\n";
# print "modified protein\n";
# print $protein,"\n";
if (length($protein)>6 && $protein!~/^\*/ && length($protein_original)>6)
{
print OUT qq!>$name-wt\n$protein_original\n!;
#$description_.=")";
#print LOG_MOD qq!>$name-indel $descriptions{$name} (MAP:$chr:$start$strand $segment_lengths $segment_starts) $description_\n$protein\n!;
print OUT_MOD qq!>$name-indel-$description_\n$protein\n!;
}
}
} else { print LOG qq!Error parsing $bed{$name}\n!; }
}
} else { print LOG qq!Error in name $chr: $line\n!; }
close(IN);
}
}
print STAT qq!
done;
!;
print STAT qq!\nnumber of modifications\tnumber of proteins\tnumber of proteins (somatic variants)\n!;
#for (my $k=0;$k<=100;$k++) { print STAT qq!$k\t$protein_modifications_distr[$k]\t$protein_modifications_distr_somatic[$k]\n!; }
close(OUT);
close(OUT_MOD);
#close(OUT_FS);
close(LOG);
close(LOG_MOD);
close(STAT);
}