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Creat.0fold-4fold.sites.pl
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#! perl
use strict;
use warnings;
use Bio::SeqIO;
my $introduction="This script is used for identify 4 fold degenerate sites from a genome with the help of gene annotation file. (Created by WangKun)";
my ($genome_file,$gff_file,$fold)=@ARGV;
die "Introduction: $introduction\nUsage: $0 <genome file> <gff file> <4 fold degeneration sites output>\n" if(@ARGV < 3);
my %gff;
open(I,"< $gff_file");
my $no=0;
while(<I>){
chomp;
next if /^$/;
next if(/^#/);
my @a=split(/\s+/);
next unless($a[2] eq "CDS");
$no++;
my ($chr,$start,$end,$strand,$phase,$name)=($a[0],$a[3],$a[4],$a[6],$a[7],$a[8]);
# $chr=~s/chr//g;
$name=~/Parent=([^;]+)/;
$name=$1;
$gff{$chr}{$name}{$no}{start}=$start;
$gff{$chr}{$name}{$no}{end}=$end;
$gff{$chr}{$name}{$no}{strand}=$strand;
$gff{$chr}{$name}{$no}{phase}=$phase;
}
close I;
print STDERR "GFF reading complete!\n";
my $fa=Bio::SeqIO->new(-file=>$genome_file,-format=>'fasta');
my $control=0;
open(O,"> $fold");
while(my $seq=$fa->next_seq){
my $chr=$seq->id;
my $seq=$seq->seq;
next unless(exists $gff{$chr});
foreach my $name(sort keys %{$gff{$chr}}){
my $strand="NA";
my $line="";
# die "debug" if($control++>100);
foreach my $no(sort { $gff{$chr}{$name}{$a}{start} <=> $gff{$chr}{$name}{$b}{start} } keys %{$gff{$chr}{$name}}){
if($strand eq "NA"){
$strand=$gff{$chr}{$name}{$no}{strand};
}
my $start=$gff{$chr}{$name}{$no}{start};
my $end=$gff{$chr}{$name}{$no}{end};
my $len=$end-$start+1;
my $subline=substr($seq,$start-1,$len);
$line.=$subline;
}
my @four_fold_sites_location_in_genome;
my @zero_fold_sites_location_in_genome;
if($strand eq "+"){
my $pep = &translate_nucl($line);
my ($four_fold_sites_location_in_cds,$zero_fold_sites_location_in_cds) = &get_location_in_cds($line);
my @four_fold_sites_location_in_cds=@{$four_fold_sites_location_in_cds};
my @zero_fold_sites_location_in_cds=@{$zero_fold_sites_location_in_cds};
next if(scalar(@four_fold_sites_location_in_cds) == 0 && scalar(@zero_fold_sites_location_in_cds)==0);
my %corresponding_postion;
my $accumulating_length=0;
foreach my $no(sort { $gff{$chr}{$name}{$a}{start} <=> $gff{$chr}{$name}{$b}{start} } keys %{$gff{$chr}{$name}}){
my $start=$gff{$chr}{$name}{$no}{start};
my $end=$gff{$chr}{$name}{$no}{end};
my $len=$end-$start+1;
my $real_start=$start;
my $real_end =$end;
my $cds_start =$accumulating_length+1;
my $cds_end =$accumulating_length+$len;
my $real_pos=$real_start;
for(my $i=$cds_start;$i<=$cds_end;$i++){
my $cds_pos=$i;
$corresponding_postion{$cds_pos}=$real_pos;
$real_pos++;
}
$accumulating_length+=$len;
}
foreach my $cds_pos(@four_fold_sites_location_in_cds){
my $real_pos=$corresponding_postion{$cds_pos};
print O "$chr\t$real_pos\t$cds_pos\t$name\tfour-fold\n";
}
foreach my $cds_pos(@zero_fold_sites_location_in_cds){
my $real_pos=$corresponding_postion{$cds_pos};
print O "$chr\t$real_pos\t$cds_pos\t$name\tzero-fold\n";
}
}
elsif($strand eq "-"){
$line=reverse($line);
$line=~tr/ATCGatcg/TAGCtagc/;
my $pep = &translate_nucl($line);
my ($four_fold_sites_location_in_cds,$zero_fold_sites_location_in_cds) = &get_location_in_cds($line);
my @four_fold_sites_location_in_cds=@{$four_fold_sites_location_in_cds};
my @zero_fold_sites_location_in_cds=@{$zero_fold_sites_location_in_cds};
next if(scalar(@four_fold_sites_location_in_cds) == 0 && scalar(@zero_fold_sites_location_in_cds)==0);
my %corresponding_postion;
my $accumulating_length=0;
foreach my $no(sort { $gff{$chr}{$name}{$b}{start} <=> $gff{$chr}{$name}{$a}{start} } keys %{$gff{$chr}{$name}}){
my $start=$gff{$chr}{$name}{$no}{start};
my $end=$gff{$chr}{$name}{$no}{end};
my $len=$end-$start+1;
my $real_start=$start;
my $real_end =$end;
my $cds_start =$accumulating_length+1;
my $cds_end =$accumulating_length+$len;
my $real_pos=$real_end;
for(my $i=$cds_start;$i<=$cds_end;$i++){
my $cds_pos=$i;
$corresponding_postion{$cds_pos}=$real_pos;
$real_pos--;
}
$accumulating_length+=$len;
}
foreach my $cds_pos(@four_fold_sites_location_in_cds){
my $real_pos=$corresponding_postion{$cds_pos};
print O "$chr\t$real_pos\t$cds_pos\t$name\tfour-fold\n";
}
foreach my $cds_pos(@zero_fold_sites_location_in_cds){
my $real_pos=$corresponding_postion{$cds_pos};
print O "$chr\t$real_pos\t$cds_pos\t$name\tzero-fold\n";
}
}
print STDERR "$chr\t$name\n";
# last;
}
# last;
}
close O;
sub get_location_in_cds{
my $line=shift;
my @location_four_fold;
my @location_zero_fold;
my $len=length($line);
if($len == 0){
# return(@location);
return(\@location_four_fold,\@location_zero_fold);
}
my @bases=split("",$line);
my @ATCG=("A","T","C","G");
for(my $i=0;$i<$len;$i+=3){
next if(!$bases[$i] or !$bases[$i+1] or !$bases[$i+2]);
my @codon_bases=($bases[$i],$bases[$i+1],$bases[$i+2]);
my $codon_bases=join "",@codon_bases;
$codon_bases=~tr/atcg/ATCG/;
next if($codon_bases=~/[^ATCG]/);
my $codon_pep = &translate_nucl($codon_bases);
for(my $j=0;$j<3;$j++){
my $light = 0;
my @new_codon_bases = @codon_bases;
my $type="NA";
foreach my $bases(@ATCG){
$new_codon_bases[$j]=$bases;
my $new_codon_bases = join "",@new_codon_bases;
next if($new_codon_bases eq $codon_bases);
my $new_codon_pep = &translate_nucl($new_codon_bases);
if($new_codon_pep ne $codon_pep){
$light++;
# last;
}
}
my $location_in_cds=$i+$j+1;
if($light==3){
$type="0";
push @location_zero_fold,$location_in_cds;
}
elsif ($light==0) {
$type="4";
push @location_four_fold,$location_in_cds;
}
}
}
my $number_four_fold=@location_four_fold;
my $number_zero_fold=@location_zero_fold;
print STDERR "$number_four_fold four-fold-degenerate-sites and $number_zero_fold zero-fold-degenerate-sites in this gene\n";
return(\@location_four_fold,\@location_zero_fold);
}
sub translate_nucl{
my $seq=shift;
my $seq_obj=Bio::Seq->new(-seq=>$seq,-alphabet=>'dna');
my $pro=$seq_obj->translate;
$pro=$pro->seq;
return($pro);
}