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peekseq.pl
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#!/usr/bin/env perl
#AUTHOR
# Rene Warren
# rwarren at bcgsc.ca
#NAME
# peekseq : Protein Estimation systEm in [DNA/RNA] SEQuences, using K-mers
#SYNOPSIS
# Identifies DNA regions with coding potential de novo, using a k-mer approach
#DOCUMENTATION
# README.md distributed with this software
# We hope this code is useful to you -- Please send comments & suggestions to rwarren * bcgsc.ca
#LICENSE
#peekseq Copyright (c) 2023 British Columbia Cancer Agency Branch. All rights reserved.
#peekseq is released under the GNU General Public License v3
use strict;
use Getopt::Std;
use vars qw($opt_k $opt_f $opt_s $opt_v $opt_c);
getopts('k:f:s:v:c:');
my $version = "v0.0.1";
my ($k,$tsvflag,$code) = (90,0,1);
my $regsz = 3 * $k;
system("clear");
if(! $opt_f){
print "Usage: $0 $version\n";
print " -f FASTA (DNA/RNA) file (required)\n";
print " -k length (option, default: -k $k)\n";
print " -c genetic code translation table id (option, default: -c $code [standard])\n";
print " -s min. reference FASTA region [size] (bp) to output (option, default: -s $regsz bp)\n";
die " -v output tsv file (option, -v 1==yes -v 0==no [default])\n\n";
}
### Fetch options
my $f = $opt_f; #reference
$k = $opt_k if($opt_k);
$regsz = $opt_s if($opt_s);
$tsvflag = $opt_v if($opt_v);
$code = $opt_c if($opt_c);
###Prepare output
#-----
my $fn = "peekseq_" . $version . "-f_" . $f . "-k" . $k;
my $tsv= $fn . "-frameKmers.tsv";
$fn .= "-s" . $regsz . "-c" . $code;
my $dna=$fn . "-codingDNA.fa";
my $pep=$fn . "-codingPROTEIN.fa";
my $log=$fn . ".log";
open(LOG,">$log") || die "Can't write to $log -- fatal.\n";
my $message = "\nRunning: $0 $version\n\t-k $k\n\t-f $f\n\t-s $regsz\n\t-c $code\n\t-v $tsvflag\n";
print $message;
print LOG $message;
###Checking files and options
#-----
if(! -e $f){
die "Invalid file: $f -- fatal\n";
}
if($k % 3 != 0){
die "-k $k must be a multiple of 3 -- fatal.\n";
}
&evalCP($f,$k,$dna,$pep,$regsz,$tsv,$code,$tsvflag);
$message = "done.\n";
$message .= "-" x 30, "\n";
$message .= "\nOutput in-frame regions >= $regsz bp in:\n$dna\n$pep\n";
$message .= "\nOutput $k-mers frames:\n$tsv\ndone.\n" if($tsvflag);
print $message;
print LOG $message;
close LOG;
exit;
#--------------------------------
sub evalCP{
my ($f,$k,$dna,$pep,$regsz,$tsv,$code,$tsvflag) = @_;
my ($head,$prevhead,$seq,$preventry) = ("","","","");
open(DNA,">$dna") || die "Can't write $dna -- fatal.\n";
open(PEP,">$pep") || die "Can't write $pep -- fatal.\n";
if($tsvflag){
open(TSV,">$tsv") || die "Can't write $tsv -- fatal.\n";
print TSV "header\tposition\tframe\tcoding\n";
}
my ($codon2aa,$startcodon) = &initializeAA($code);
###Support for compressed files
if($f=~/zip$/i){
open(IN,"unzip -p $f|") || die "Error reading $f -- fatal\n";
}elsif($f=~/gz$/i || $f=~/gzip$/i){
open(IN,"gunzip -c $f|") || die "Error reading $f -- fatal\n";
}else{
open(IN,$f) || die "Error reading $f -- fatal\n";
}
while(<IN>){
s/\r\n/\n/g;### DOS to UNIX
chomp;
if(/^\>(\S+)/){
my $entry = $1;
$head = $entry;#$_;
if($prevhead ne $head && $prevhead ne "" && $seq ne ""){
&printOutput($codon2aa,$startcodon,$prevhead,$seq,$preventry,$k,$regsz,$tsvflag);
}
$seq = "";
$prevhead = $head;
$preventry = $entry;
}else{
my $seqstretch = $1 if(/^(\S+)/); ###this prevents DOS new lines from messing up the TSV output
$seq .= uc($seqstretch);
}
}
&printOutput($codon2aa,$startcodon,$prevhead,$seq,$preventry,$k,$regsz,$tsvflag);
close DNA;
close PEP;
close TSV if($tsvflag);
}
#--------------------------------
sub printOutput{
my ($codon2aa,$startcodon,$head,$seq,$preventry,$k,$regsz,$tsvflag) = @_;
my ($initial,$unique,$notunique,$sum,$sumout,$buffer) = (-1,0,0,0,0,0);
my $s;
my $lastframe = 0;
my $lastframerev = 0;
my $lastcodon = "";
for(my $pos=0;$pos<=(length($seq)-$k);$pos++){
my $kmer = substr($seq,$pos,$k);
my $codon = substr($kmer,($k-3),3);
$lastcodon = $codon;
$kmer =~ tr/U/T/; ### handles RNA U>>>T
my $rckmer = &reverseComplement($kmer);
my $frame = ($pos % 3) + 1;
my $framerev = -1 * $frame;
$lastframe = $frame;
$lastframerev = $framerev;
my $aamer = &dna2protein($kmer,$codon2aa);
my $rcaamer = &dna2protein($rckmer,$codon2aa);
my $bit=1;
if($aamer=~/\*/ || $aamer=~/X/){# stop or end
$bit=0;
if(length($s->{$frame}{'seq'}) >= $regsz){
$s->{$frame}{'seq'} .= $codon;
$s->{$frame}{'end'} = $pos + $k;
###
my $lcdna = lc($s->{$frame}{'seq'});
my @ntarr = ( $lcdna =~ m/.../g );
my $flag=0;
my $newnt="";
my $newaa="";
foreach my $cd (@ntarr){
my $aa = lc(&dna2protein($cd,$codon2aa));
if(defined $startcodon->{$cd}){$flag=1;}
if($flag){
$newnt .= uc($cd);
$newaa .= uc($aa);
}else{
$newnt .= $cd;
$newaa .= $aa;
}
}
###
my $ntlen = length($newnt);
print DNA ">$head $s->{$frame}{'start'}-$s->{$frame}{'end'} length:$ntlen frame:$frame\n$newnt\n";
my $aalen = length($newaa);
print PEP ">$head $s->{$frame}{'start'}-$s->{$frame}{'end'} length:$aalen frame:$frame\n$newaa\n";
}
$s->{$frame}{'start'} = 0;
$s->{$frame}{'seq'} = "";
}else{
if($s->{$frame}{'seq'} eq ""){
$s->{$frame}{'seq'} = $kmer;
$s->{$frame}{'start'} = $pos;
}else{
$s->{$frame}{'seq'} .= $codon;
$s->{$frame}{'end'} = $pos + $k;
}##
}
my $rcbit=-1;
if($rcaamer=~/\*/ || $rcaamer=~/X/){###stop or end
$rcbit=0;
if(length($s->{$framerev}{'seq'}) >= $regsz){
my $rc = reverseComplement($s->{$framerev}{'seq'});
my $lcdna = lc($rc);
my @ntarr = ( $lcdna =~ m/.../g );
my $flag=0;
my $newnt="";
my $newaa="";
foreach my $cd (@ntarr){
my $aa = lc(&dna2protein($cd,$codon2aa));
if(defined $startcodon->{$cd}){$flag=1;}
if($flag){
$newnt .= uc($cd);
$newaa .= uc($aa);
}else{
$newnt .= $cd;
$newaa .= $aa;
}
}
my $ntlen = length($newnt);
print DNA ">$head $s->{$framerev}{'end'}-$s->{$framerev}{'start'} length:$ntlen frame:$framerev\n$newnt\n";
my $aalen = length($newaa);
print PEP ">$head $s->{$framerev}{'end'}-$s->{$framerev}{'start'} length:$aalen frame:$framerev\n$newaa\n";
}
$s->{$framerev}{'start'} = $pos;
$s->{$framerev}{'seq'} = $kmer;
}else{
if($s->{$framerev}{'seq'} eq ""){
$s->{$framerev}{'seq'} = $kmer;
$s->{$framerev}{'start'} = $pos;
}else{
$s->{$framerev}{'seq'} .= $codon;
$s->{$framerev}{'end'} = $pos + $k;
}
}
print TSV "$head\t$pos\t$frame\t$bit\n$head\t$pos\t$framerev\t$rcbit\n" if($tsvflag);
}###end of sequence
###print what is in the buffer (like a stop, for all 3 frames)
my @frames = (1,2,3);
foreach my $fr(@frames){
if(length($s->{$fr}{'seq'}) >= $regsz){
#$s->{$lastframe}{'seq'} .= $lastcodon;
#$s->{$lastframe}{'end'} = length($seq);
###
my $lcdna = lc($s->{$fr}{'seq'});
my @ntarr = ( $lcdna =~ m/.../g );
my $flag=0;
my $newnt="";
my $newaa="";
foreach my $cd (@ntarr){
my $aa = lc(&dna2protein($cd,$codon2aa));
if(defined $startcodon->{$cd}){$flag=1;}
if($flag){
$newnt .= uc($cd);
$newaa .= uc($aa);
}else{
$newnt .= $cd;
$newaa .= $aa;
}
}
###
my $ntlen = length($newnt);
print DNA ">$head $s->{$fr}{'start'}-$s->{$fr}{'end'} length:$ntlen frame:$fr\n$newnt\n";
my $aalen = length($newaa);
print PEP ">$head $s->{$fr}{'start'}-$s->{$fr}{'end'} length:$aalen frame:$fr\n$newaa\n";
}#length >= user-defined threshold
}#3 frames
###handle reverse
my @framesrec=(-1,-2,-3);
foreach my $fr(@framesrec){
if(length($s->{$fr}{'seq'}) >= $regsz){
my $rc = reverseComplement($s->{$fr}{'seq'});
my $lcdna = lc($rc);
my @ntarr = ( $lcdna =~ m/.../g );
my $flag=0;
my $newnt="";
my $newaa="";
foreach my $cd (@ntarr){
my $aa = lc(&dna2protein($cd,$codon2aa));
if(defined $startcodon->{$cd}){$flag=1;}
if($flag){
$newnt .= uc($cd);
$newaa .= uc($aa);
}else{
$newnt .= $cd;
$newaa .= $aa;
}
}
my $ntlen = length($newnt);
print DNA ">$head $s->{$fr}{'end'}-$s->{$fr}{'start'} length:$ntlen frame:$fr\n$newnt\n";
my $aalen = length($newaa);
print PEP ">$head $s->{$fr}{'end'}-$s->{$fr}{'start'} length:$aalen frame:$fr\n$newaa\n";
}### length>= user-defined threshold
}### all 3 frames rev
}
#--------------------------------
sub initializeAA{
my $code=shift;
my $codon2aa;
my $startcodon;
$codon2aa->{"ttt"}= "F"; $codon2aa->{"ttc"}= "F"; $codon2aa->{"tta"}="L";
$codon2aa->{"ttg"}= "L"; $codon2aa->{"tct"}= "S"; $codon2aa->{"tcc"}= "S";
$codon2aa->{"tca"}= "S"; $codon2aa->{"tcg"}= "S"; $codon2aa->{"tat"}= "Y";
$codon2aa->{"tac"}= "Y"; $codon2aa->{"tgt"}= "C"; $codon2aa->{"tgc"}= "C";
$codon2aa->{"ctt"}= "L"; $codon2aa->{"ctc"}= "L"; $codon2aa->{"cta"}= "L";
$codon2aa->{"ctg"}= "L"; $codon2aa->{"cct"}= "P"; $codon2aa->{"ccc"}= "P";
$codon2aa->{"cca"}= "P"; $codon2aa->{"ccg"}= "P"; $codon2aa->{"cat"}= "H";
$codon2aa->{"cac"}= "H"; $codon2aa->{"caa"}= "Q"; $codon2aa->{"cag"}= "Q";
$codon2aa->{"cgt"}= "R"; $codon2aa->{"cgc"}= "R"; $codon2aa->{"cga"}= "R";
$codon2aa->{"cgg"}= "R"; $codon2aa->{"att"}= "I"; $codon2aa->{"atc"}= "I";
$codon2aa->{"ata"}= "I"; $codon2aa->{"atg"}= "M"; $codon2aa->{"act"}= "T";
$codon2aa->{"acc"}= "T"; $codon2aa->{"aca"}= "T"; $codon2aa->{"acg"}= "T";
$codon2aa->{"aat"}= "N"; $codon2aa->{"aac"}= "N"; $codon2aa->{"aaa"}= "K";
$codon2aa->{"aag"}= "K"; $codon2aa->{"agt"}= "S"; $codon2aa->{"agc"}= "S";
$codon2aa->{"aga"}= "R"; $codon2aa->{"agg"}= "R"; $codon2aa->{"gtt"}= "V";
$codon2aa->{"gtc"}= "V"; $codon2aa->{"gta"}= "V"; $codon2aa->{"gtg"}= "V";
$codon2aa->{"gct"}= "A"; $codon2aa->{"gcc"}= "A"; $codon2aa->{"gca"}= "A";
$codon2aa->{"gcg"}= "A"; $codon2aa->{"gat"}= "D"; $codon2aa->{"gac"}= "D";
$codon2aa->{"gaa"}= "E"; $codon2aa->{"gag"}= "E"; $codon2aa->{"ggt"}= "G";
$codon2aa->{"ggc"}= "G"; $codon2aa->{"gga"}= "G"; $codon2aa->{"ggg"}= "G";
$codon2aa->{"tgg"}= "W";
$codon2aa->{"tag"}= "*";
$codon2aa->{"tga"}= "*";
$codon2aa->{"taa"}= "*";
$startcodon->{"atg"} = "M";
#The standard code currently allows initiation from UUG and CUG in addition to AUG.
$startcodon->{"ttg"} = "L";
$startcodon->{"ctg"} = "L";
$startcodon->{"gtg"} = "V";
#There are Differences from the Standard Code
#https://www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi?mode=c#top
#1. The Standard Code
#2. The Vertebrate Mitochondrial Code
#3. The Yeast Mitochondrial Code
#4. The Mold, Protozoan, and Coelenterate Mitochondrial Code and the Mycoplasma/Spiroplasma Code
#5. The Invertebrate Mitochondrial Code
#6. The Ciliate, Dasycladacean and Hexamita Nuclear Code
#9. The Echinoderm and Flatworm Mitochondrial Code
#10. The Euplotid Nuclear Code
#11. The Bacterial, Archaeal and Plant Plastid Code
#12. The Alternative Yeast Nuclear Code
#13. The Ascidian Mitochondrial Code
#14. The Alternative Flatworm Mitochondrial Code
#16. Chlorophycean Mitochondrial Code
#21. Trematode Mitochondrial Code
#22. Scenedesmus obliquus Mitochondrial Code
#23. Thraustochytrium Mitochondrial Code
#24. Rhabdopleuridae Mitochondrial Code
#25. Candidate Division SR1 and Gracilibacteria Code
#26. Pachysolen tannophilus Nuclear Code
#27. Karyorelict Nuclear Code
#28. Condylostoma Nuclear Code
#29. Mesodinium Nuclear Code
#30. Peritrich Nuclear Code
#31. Blastocrithidia Nuclear Code
#33. Cephalodiscidae Mitochondrial UAA-Tyr Code
if($code==2){
$codon2aa->{"aga"}="*";
$codon2aa->{"agg"}="*";
$codon2aa->{"ata"}="M";
$codon2aa->{"tga"}="W";
$startcodon->{"ata"}="M";
$startcodon->{"att"}="I";
$startcodon->{"atc"}="I";
}elsif($code==3){
$codon2aa->{"ata"}="M";
$codon2aa->{"ctt"}="T";
$codon2aa->{"ctc"}="T";
$codon2aa->{"cta"}="T";
$codon2aa->{"ctg"}="T";
$codon2aa->{"tga"}="W";
$startcodon->{"gtg"}="V";
}elsif($code==4){
$codon2aa->{"tga"}="W";
}elsif($code==5){
$codon2aa->{"tga"}="W";
$codon2aa->{"ata"}="M";
$codon2aa->{"agg"}="S";
$codon2aa->{"aga"}="S";
$startcodon->{"ata"}="M";
$startcodon->{"att"}="I";
}elsif($code==6){
$codon2aa->{"taa"}="Q";
$codon2aa->{"tag"}="Q";
}elsif($code==9){
$codon2aa->{"aaa"}="N";
$codon2aa->{"aga"}="S";
$codon2aa->{"agg"}="S";
$codon2aa->{"tga"}="W";
$startcodon->{"gtg"}="V";
$startcodon->{"ttg"}="L";
}elsif($code==10){
$codon2aa->{"tga"}="C";
}elsif($code==12){
$codon2aa->{"ctg"}="S";
}elsif($code==13){
$codon2aa->{"aga"}="G";
$codon2aa->{"agg"}="G";
$codon2aa->{"ata"}="M";
$codon2aa->{"tga"}="W";
$startcodon->{"gtg"}="V";
$startcodon->{"ttg"}="L";
$startcodon->{"ata"}="M";
}elsif($code==14){
$codon2aa->{"aaa"}="N";
$codon2aa->{"aga"}="S";
$codon2aa->{"agg"}="S";
$codon2aa->{"taa"}="Y";
$codon2aa->{"tga"}="W";
}elsif($code==16){
$codon2aa->{"tag"}="L";
}elsif($code==21){
$codon2aa->{"tga"}="W";
$codon2aa->{"ata"}="M";
$codon2aa->{"aga"}="S";
$codon2aa->{"agg"}="S";
$codon2aa->{"aaa"}="N";
}elsif($code==22){
$codon2aa->{"tca"}="*";
$codon2aa->{"tag"}="L";
}elsif($code==24){
$codon2aa->{"aga"}="S";
$codon2aa->{"agg"}="K";
$codon2aa->{"tga"}="W";
}elsif($code==25){
$codon2aa->{"tga"}="G";
$startcodon->{"gtg"}="V";
$startcodon->{"ttg"}="L";
}elsif($code==26){
$codon2aa->{"ctg"}="A";
$startcodon->{"gtg"}="V";
$startcodon->{"ttg"}="L";
}elsif($code==27){
$codon2aa->{"tag"}="Q";
$codon2aa->{"taa"}="Q";
#or $codon2aa->{"tga"}="W";
}elsif($code==28){
#or$codon2aa->{"tag"}="Q";
#or $codon2aa->{"taa"}="Q";
#or $codon2aa->{"tga"}="W";
}elsif($code==29){
$codon2aa->{"tag"}="Y";
$codon2aa->{"taa"}="Y";
}elsif($code==30){
$codon2aa->{"tag"}="E";
$codon2aa->{"taa"}="E";
}elsif($code==31){
$codon2aa->{"tga"}="W";
#or $codon2aa->{"tag"}="E";
#or $codon2aa->{"taa"}="E";
}elsif($code==33){
$codon2aa->{"aga"}="S";
$codon2aa->{"taa"}="Y";
$codon2aa->{"tga"}="W";
$codon2aa->{"agg"}="K";
}
return $codon2aa,$startcodon;
}
#--------------------------------
sub dna2protein{
my ($seq,$codon2aa) = @_;
my $seq = lc($seq);
my @codonlist = ( $seq =~ m/.../g );
my $aa_seq = "";
foreach my $codon (@codonlist){
#print ">>>$codon<<<\n";
if (defined $codon2aa->{$codon}){
$aa_seq.=$codon2aa->{$codon};
}else{
$aa_seq.="X";
}
}
return $aa_seq;
}
#--------------------------------
sub reverseComplement{
$_ = shift;
$_ = uc();
tr/ATGCYRKMBDHV/TACGRYMKVHDB/;
return (reverse());
}