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anablast_v1.1.pl
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anablast_v1.1.pl
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#!/usr/bin/perl -w
use strict;
$|++;
sub parse_fasta {
################################################################################
# Obtiene la longitud y la secuencia de las secuencias de un multifasta, crea
# un fichero que puede ser utilizado por el script wigtoBigWig y devuelve un
# hash con la longitud y la secuencia sin saltos de línea
# ejemplo: &parse_fasta(<path_to_fasta> <workdir>)
################################################################################
my $fasta = shift;
my $workdir = shift;
print STDERR "\nExtracting Chromosones length\n";
my %seq;
my $id = "no_id";
open FASTA, $fasta or die $!;
while (my $line = <FASTA>) {
chomp $line;
$line =~ s/[\n\r]+$//g;
next if ($line =~ m/^$/);
if ($line =~ m/^>([^\s\t\n]+)/) {
$id = $1;
next;
}
else {
$line = uc($line);
$seq{$id}{nn} .= $line;
$seq{$id}{length} += length($line);
}
}
close FASTA or die $!;
open SIZE, ">$workdir/chrom.size" or die $!;
print STDERR "Chromosome sizes:\n";
for my $key (keys %seq) {
print STDERR "$key\t$seq{$key}{length}\n";
print SIZE "$key\t$seq{$key}{length}\n";
}
close SIZE or die $!;
print STDERR "chrom.size created\n";
return \%seq;
}
sub bitscore_filter_frame_split {
################################################################################
# filtra los resultados del blast quedandose solo con los que tienen un bitscore
# de 30 o superior y elimina las repeticiones
# ejemplo: &bitscore_filter(<path_to_blast> <workdir> <bit_threshold> <ref_seq>)
################################################################################
my ($blast, $workdir, $bit_threshold, $ref_seq) = @_;
print STDERR "\nFiltering Blast by Bitscore >= $bit_threshold and spliting by frame...\n";
open BLAST, $blast or die $!;
my %path = (
1 => "$workdir/frame1.blast",
2 =>"$workdir/frame2.blast",
3 =>"$workdir/frame3.blast",
-1 => "$workdir/frame-1.blast",
-2 => "$workdir/frame-2.blast",
-3 =>"$workdir/frame-3.blast"
);
my %count;
my %file;
my @frame = (1, 2, 3, -1, -2, -3);
foreach (@frame) {
open $file{$_}, ">$path{$_}" or die $!;
}
my $prev_line = "";
while (my $line = <BLAST>) {
next if ($prev_line eq $line); # Elimina las líneas repetidas consecutivas
$prev_line = $line;
my ($seqid, $chrom, $start, $end, $evalue, $bit, $frame) = split (/\t/, $line);
my $f = 0;
for my $key (keys %{$ref_seq}) {
if ($key eq $chrom) {
$f = 1;
last;
}
}
if ($f == 0) {
print STDERR "Blast warning: no chrom id ($chrom) in seq file\n";
next;
}
if ($bit >= $bit_threshold) {
foreach (@frame) {
if ($frame == $_) {
$count{$_}++;
print {$file{$_}} $line;
last;
}
}
}
}
close BLAST or die $!;
foreach (@frame) {
close $file{$_} or die $!;
}
if (!%count) {
print STDERR "No blast hit with bitscore higher than $bit_threshold\n"
}
foreach (@frame) {
if ($count{$_}) {
print STDERR "$count{$_} lines keeped in frame $_\n";
}
else {
print STDERR "0 lines keeped in frame $_\n";
}
}
return %path;
}
sub frame_to_wig_and_bigwig {
################################################################################
# Analiza los resultados del blast según el frame que le indiques y te crea un
# fichero .wig con los picos de anablas para ese frame.
# necesita pasar el blast por la subrutina bitscore_filter_frame_split previamente
# para que funcione correctamente y que la subrutina fasta_length cree el fichero
# chrom.size
# ejemplo: &parse_frame(<frame>, <blast.result>)
################################################################################
my ($frame, $blast, $workdir) = @_;
my %chr;
my $wig_path = "$workdir/frame$frame.wig";
print STDERR "\nparsing frame: $frame\n";
open BLAST, $blast or die $!;
if(-z $blast) {
print STDERR "Empty blast report\n";
open OUT, ">$wig_path" or die $!;
print OUT "track type=wiggle_0 name=\"frame$frame\" description=\"frame$frame\" visibility=full graphType=bar\n";
print STDERR "Empty wig created. Next\n";
}
else {
while(<BLAST>){
chomp $_;
my @colums = split (/\t/, $_);
if ($colums[2] < $colums[3]) {
for(my $i = $colums[2]; $i <= $colums[3]; $i++){
$chr{$colums[1]}[$i]++;
}
}
else {
for(my $i = $colums[3]; $i <= $colums[2]; $i++){
$chr{$colums[1]}[$i]++;
}
}
}
close BLAST;
open OUT, ">$wig_path" or die $!;
print OUT "track type=wiggle_0 name=\"frame$frame\" description=\"frame$frame\" visibility=full graphType=bar\n";
foreach my $key(sort keys %chr){
print OUT "variableStep chrom=$key\n";
for my $i(1 .. $#{$chr{$key}}){
if(!$chr{$key}[$i]){
print OUT "$i\t0\n";
next;
}
print OUT "$i\t$chr{$key}[$i]\n";
}
}
close OUT;
print STDERR "frame$frame.wig created\n";
print STDERR "Creating BigWig...\n";
`./wigToBigWig $workdir/frame$frame.wig $workdir/chrom.size $workdir/frame$frame.bw`;
print STDERR "BigWig Created\n";
}
return $wig_path;
}
sub reverse_comp {
################################################################################
# Obtiene la reversa complementaria de una secuencia dada
# ejemplo: &reverse_comp(<secuenca de DNA>)
################################################################################
my $seq = shift;
$seq =~ tr/acgtACGT/tgcaTGCA/;
$seq = reverse $seq;
return $seq;
}
sub dna2aa {
################################################################################
# Traduce una secuencia de ADN a proteínas, se le debe indicar en que fase se
# encuentra (1, 2, 3)
# ejemplo: &dna2aa(<DNA_seq>, <frame>)
################################################################################
#código genético
my %genetic_code = (
'TCA' => 'S', # Serine
'TCC' => 'S', # Serine
'TCG' => 'S', # Serine
'TCT' => 'S', # Serine
'TTC' => 'F', # Phenylalanine
'TTT' => 'F', # Phenylalanine
'TTA' => 'L', # Leucine
'TTG' => 'L', # Leucine
'TAC' => 'Y', # Tyrosine
'TAT' => 'Y', # Tyrosine
'TAA' => '*', # Stop
'TAG' => '*', # Stop
'TGC' => 'C', # Cysteine
'TGT' => 'C', # Cysteine
'TGA' => '*', # Stop
'TGG' => 'W', # Tryptophan
'CTA' => 'L', # Leucine
'CTC' => 'L', # Leucine
'CTG' => 'L', # Leucine
'CTT' => 'L', # Leucine
'CCA' => 'P', # Proline
'CCC' => 'P', # Proline
'CCG' => 'P', # Proline
'CCT' => 'P', # Proline
'CAC' => 'H', # Histidine
'CAT' => 'H', # Histidine
'CAA' => 'Q', # Glutamine
'CAG' => 'Q', # Glutamine
'CGA' => 'R', # Arginine
'CGC' => 'R', # Arginine
'CGG' => 'R', # Arginine
'CGT' => 'R', # Arginine
'ATA' => 'I', # Isoleucine
'ATC' => 'I', # Isoleucine
'ATT' => 'I', # Isoleucine
'ATG' => 'M', # Methionine
'ACA' => 'T', # Threonine
'ACC' => 'T', # Threonine
'ACG' => 'T', # Threonine
'ACT' => 'T', # Threonine
'AAC' => 'N', # Asparagine
'AAT' => 'N', # Asparagine
'AAA' => 'K', # Lysine
'AAG' => 'K', # Lysine
'AGC' => 'S', # Serine
'AGT' => 'S', # Serine
'AGA' => 'R', # Arginine
'AGG' => 'R', # Arginine
'GTA' => 'V', # Valine
'GTC' => 'V', # Valine
'GTG' => 'V', # Valine
'GTT' => 'V', # Valine
'GCA' => 'A', # Alanine
'GCC' => 'A', # Alanine
'GCG' => 'A', # Alanine
'GCT' => 'A', # Alanine
'GAC' => 'D', # Aspartic Acid
'GAT' => 'D', # Aspartic Acid
'GAA' => 'E', # Glutamic Acid
'GAG' => 'E', # Glutamic Acid
'GGA' => 'G', # Glycine
'GGC' => 'G', # Glycine
'GGG' => 'G', # Glycine
'GGT' => 'G', # Glycine
);
my $prot;
my $dna = shift;
my $frame = shift;
my $length = length($dna);
my $res = $length%3;
my $c = 0;
for(my $x=$frame-1; $x < $length - $res; $x += 3){
my $codon = substr($dna,$x,3);
$codon = uc($codon);
if (!$genetic_code{$codon}) {
$prot .= "?";
$c++;
#print STDERR "Warning: Extrange nucleotide ($codon) detected in dna2aa subrutine. AA ? created\n";
next;
}
$prot .= $genetic_code{$codon};
}
if ($c != 0) {
print STDERR "Warning: $c \"?\" Amino acids created\n";
}
return $prot;
}
sub frame_calc {
################################################################################
# Calcula el frame de una subsecuencia a partir del frame original de la
# secuencia original teniendo en cuenta la reversa complementaria
# ejemplo: &frame_calc(<frame>, <start>, <end>, <sequence_length>)
################################################################################
my $ori_frame = shift;
my $start = shift;
my $end = shift;
my $seq_length = shift;
my $frame;
my $res;
if ($ori_frame > 0) {
$res = ($start -1) % 3;
if($res == 0){
$frame = $ori_frame;
}
elsif($res == 1){
if($ori_frame == 1){
$frame = 3;
}
elsif($ori_frame == 2){
$frame = 1;
}
elsif($ori_frame == 3){
$frame = 2;
}
}
elsif($res == 2){
if($ori_frame == 1){
$frame = 2;
}
elsif($ori_frame == 2){
$frame = 3;
}
elsif($ori_frame == 3){
$frame = 1;
}
}
}
else {
$res = ($seq_length - $end) % 3;
if($res == 0){
if($ori_frame == -1){
$frame = 1;
}
elsif($ori_frame == -2){
$frame = 2;
}
elsif($ori_frame == -3){
$frame = 3;
}
}
elsif($res == 1){
if($ori_frame == -1){
$frame = 3;
}
elsif($ori_frame == -2){
$frame = 1;
}
elsif($ori_frame == -3){
$frame = 2;
}
}
elsif($res == 2){
if($ori_frame == -1){
$frame = 2;
}
elsif($ori_frame == -2){
$frame = 3;
}
elsif($ori_frame == -3){
$frame = 1;
}
}
}
# print "\n$ori_frame\t($res) => $frame\n";
return $frame;
}
sub extract_peaks {
################################################################################
# Extrae los datos de los picos de un marco de lectura determinado con unos
# determinados valores desde un fichero WIG
# Se puede modificar la altura mínima del pico y desde que altura se empieza o se
# deja de condiderar que es un pico
# ejemplo: &extract_pic(<path_to_wig>, <sequence_reference>, <blast_index_reference>)
################################################################################
my ($wig, $seq_ref) = @_; # Se ha eliminado el tercer argumento $blat_ref para eliminar el cáculo de pvalor
my %seq = %{$seq_ref};
print STDERR "Extracting peaks from WIG: $wig\n";
my $chrom;
my $frame;
my $start;
my $end;
my $f = 0;
my $f2 = 0;
my %pic;
my $top = 0;
my $c = 0;
my $position;
open WIG, "$wig" or die $!;
while(<WIG>){
chomp;
if($_ =~ m/variableStep chrom=(.+)/){
if($f == 1 and $f2 == 1) {
$f = 0;
$f2 = 0;
$c++;
$end = $position;
$pic{"frame_$frame"."_peak_$c"}{frame} = $frame;
$pic{"frame_$frame"."_peak_$c"}{chrom} = $chrom;
$pic{"frame_$frame"."_peak_$c"}{start} = $start;
$pic{"frame_$frame"."_peak_$c"}{end} = $end;
$pic{"frame_$frame"."_peak_$c"}{position} = "$chrom:$start..$end";
$pic{"frame_$frame"."_peak_$c"}{top} = $top;
$pic{"frame_$frame"."_peak_$c"}{length} = $end - $start + 1;
if ($frame >= 1) {
$pic{"frame_$frame"."_peak_$c"}{strand} = "+";
}
else {
$pic{"frame_$frame"."_peak_$c"}{strand} = "-";
}
# $pic{"frame_$frame"."_peak_$c"}{pvalue} = &pvalue_calc($chrom, $frame, $start, $end, $blast_ref); # comentado para eliminar el calculo de pvalor
# my $p = $pic{"frame_$frame"."_peak_$c"}{pvalue};
# print "$chrom:$start..$end\t$p\n";
# exit if ($p == 1);
$top = 0;
}
$chrom = $1;
next;
}
elsif ($_ =~ m/track type=wiggle_0 name=\"frame(.+)\" description=\"frame/) {
$frame = $1;
next;
}
next unless($_ =~ m/^(\d+)\t(\d+)$/);
$position = $1;
if($2 >= 15 and $f == 0){
$start = $1;
$f = 1;
}
if($2 >= 20){ #Altura mínima para considerarlo un pico
$f2 = 1;
if($2 > $top){
$top = $2;
}
}
elsif($2 <= 15 and $f == 1){
$end = $1;
$f = 0;
# print "f$frame\ts$start\tt$top\te$end\n"; ## prueba para ver donde comienzan y terminan los picos
if($f2 == 1){
$f2 = 0;
$c++;
$pic{"frame_$frame"."_peak_$c"}{frame} = $frame;
$pic{"frame_$frame"."_peak_$c"}{chrom} = $chrom;
$pic{"frame_$frame"."_peak_$c"}{start} = $start;
$pic{"frame_$frame"."_peak_$c"}{end} = $end;
$pic{"frame_$frame"."_peak_$c"}{position} = "$chrom:$start..$end";
$pic{"frame_$frame"."_peak_$c"}{top} = $top;
$pic{"frame_$frame"."_peak_$c"}{length} = $end - $start + 1;
if ($frame >= 1) {
$pic{"frame_$frame"."_peak_$c"}{strand} = "+";
}
else {
$pic{"frame_$frame"."_peak_$c"}{strand} = "-";
}
# $pic{"frame_$frame"."_peak_$c"}{pvalue} = &pvalue_calc($chrom, $frame, $start, $end, $blast_ref); # comentado para eliminar el calculo de pvalor
# my $p = $pic{"frame_$frame"."_peak_$c"}{pvalue};
# print "$chrom:$start..$end\t$p\n";
# exit if ($p == 1);
$top = 0;
}
}
}
if ($f == 1 and $f2 == 1) {
$f = 0;
$f2 = 0;
$c++;
$end = $position;
$pic{"frame_$frame"."_peak_$c"}{frame} = $frame;
$pic{"frame_$frame"."_peak_$c"}{chrom} = $chrom;
$pic{"frame_$frame"."_peak_$c"}{start} = $start;
$pic{"frame_$frame"."_peak_$c"}{end} = $end;
$pic{"frame_$frame"."_peak_$c"}{position} = "$chrom:$start..$end";
$pic{"frame_$frame"."_peak_$c"}{top} = $top;
$pic{"frame_$frame"."_peak_$c"}{length} = $end - $start + 1;
if ($frame >= 1) {
$pic{"frame_$frame"."_peak_$c"}{strand} = "+";
}
else {
$pic{"frame_$frame"."_peak_$c"}{strand} = "-";
}
# $pic{"frame_$frame"."_peak_$c"}{pvalue} = &pvalue_calc($chrom, $frame, $start, $end, $blast_ref); Comentado para eliminar el calculo del pvalor
# my $p = $pic{"frame_$frame"."_peak_$c"}{pvalue};
# print "$chrom:$start..$end\t$p\n";
# exit if ($p == 1);
}
close WIG or die $!;
print STDERR "Total peaks in $wig = $c\n";
print STDERR "Extracting $wig peak sequences\n";
foreach my $key (keys %pic) {
$pic{$key}{nn} = substr($seq{$pic{$key}{chrom}}{nn}, $pic{$key}{start} -1, $pic{$key}{end} - $pic{$key}{start} +1);
$pic{$key}{nn} = uc($pic{$key}{nn});
# print STDERR "$key:$pic{$key}{start}..$pic{$key}{end}\n$pic{$key}{nn}\n"; ## prueba para ver como se ve la secuencia
if ($pic{$key}{frame} < 0) {
$pic{$key}{nn} = &reverse_comp($pic{$key}{nn});
}
$pic{$key}{aa} = &dna2aa($pic{$key}{nn}, &frame_calc($pic{$key}{frame}, $pic{$key}{start}, $pic{$key}{end}, $seq{$pic{$key}{chrom}}{length}));
my @aa = split ("", $pic{$key}{aa});
my $stop_codon = 0;
foreach my $a (@aa) {
if ($a eq "*") {
$stop_codon++;
}
}
$pic{$key}{stop_codon} = $stop_codon;
}
return \%pic;
}
sub pvalue_calc {
my ($chr, $frame, $start, $end, $ref_blast_index) = @_;
my %blast_index = %{$ref_blast_index};
my $pvalue = 1;
my $start_range = 10000 * (int($start/10000) + 1);
my $end_range = 10000 * (int($end/10000) + 1);
my %nn;
my $length = $end - $start + 1;
for my $n ($start..$end) {
$nn{$n} = 1;
}
for (my $i = $start_range; $i <= $end_range; $i += 10000) {
foreach my $k (keys %{$blast_index{$chr}{$frame}{$i}}) {
my $b_uniref = $blast_index{$chr}{$frame}{$i}{$k}{uniref};
my $b_evalue = $blast_index{$chr}{$frame}{$i}{$k}{evalue};
my $b_start = $blast_index{$chr}{$frame}{$i}{$k}{start};
my $b_end = $blast_index{$chr}{$frame}{$i}{$k}{end};
my $b_pvalue = $blast_index{$chr}{$frame}{$i}{$k}{pvalue};
my $b_length = $blast_index{$chr}{$frame}{$i}{$k}{length};
next if ($start > $b_end or $end < $b_start);
my $overlap;
for my $n ($b_start..$b_end) {
if ($nn{$n}) {
$overlap++;
}
}
next unless ($overlap/$length >= 0.2 or $overlap/$b_length >= 0.2);
#print "\t$b_uniref\t$b_start\t$b_end\t$b_evalue\t$b_pvalue\n";
$pvalue *= $b_pvalue;
}
}
return $pvalue;
}
sub blast_index {
################################################################################
# Parte de un fichero en formato blast y lo indexa en memoria para poder trabajar
# con el devolviendo la referencia de un hash de hashes
# ejemplo: &gff_index(<path to blast>)
################################################################################
my ($path_to_blast) = @_;
print STDERR "\nIndexing blast $path_to_blast\n";
my %hash;
my $c;
open BLAST, $path_to_blast or die $!;
while (<BLAST>) {
chomp;
$c++;
next if ($_ =~ m/^#/ or $_ =~ m/^$/);
my ($uniref, $chr, $start, $end, $evalue, $bit, $frame) = split (/\t/,$_);
if ($start > $end) {
my $s_temp = $start;
my $e_temp = $end;
$start = $e_temp;
$end = $s_temp;
}
my $e = 2.718281828459045235360;
my $exp= $e**(-$evalue);
my $pvalue= 1-$exp;
my $start_range = 10000 * (int($start/10000) + 1);
my $end_range = 10000 * (int($end/10000) + 1);
for (my $i = $start_range; $i <= $end_range; $i += 10000) {
$hash{$chr}{$frame}{$i}{$c}{evalue} = $evalue;
$hash{$chr}{$frame}{$i}{$c}{uniref} = $uniref;
$hash{$chr}{$frame}{$i}{$c}{chr} = $chr;
$hash{$chr}{$frame}{$i}{$c}{start} = $start;
$hash{$chr}{$frame}{$i}{$c}{end} = $end;
$hash{$chr}{$frame}{$i}{$c}{frame} = $frame;
$hash{$chr}{$frame}{$i}{$c}{pvalue} = $pvalue;
$hash{$chr}{$frame}{$i}{$c}{length} = $end - $start + 1;
}
}
close BLAST or die $!;
return \%hash;
}
sub peaks_to_tsv {
################################################################################
# Guarda los picos en un fichero de texto en formato tsv
# ejemplo: &peaks_to_tsv(<ref_peaks>, <path_to_tsv>)
################################################################################
print STDERR "\nPrinting peaks in tsv\n";
my $ref_peaks = shift;
my %peaks = %{$ref_peaks};
my $tsv = shift;
open OUT, ">$tsv" or die $!;
foreach my $key (keys %peaks) {
print OUT "id\t";
foreach my $key2 (sort keys %{$peaks{$key}}) {
print OUT "$key2\t";
}
print OUT "\n";
last;
}
foreach my $key (keys %peaks) {
print OUT "$key\t";
foreach my $key2 (sort keys %{$peaks{$key}}) {
print OUT "$peaks{$key}{$key2}\t";
}
print OUT "\n";
}
close OUT or die $!;
}
sub tsv_to_peaks {
my $tsv = shift;
my %peaks;
my @header;
print STDERR "Importing peaks from $tsv\n";
open TSV, "$tsv" or die $!;
while (<TSV>) {
if ($_ =~ m/^id/) {
@header = split(/\t/, $_);
chomp @header;
next;
}
my @data = split(/\t/, $_);
chomp @data;
foreach my $i (1..$#header) {
$peaks{$data[0]}{$header[$i]} = $data[$i];
}
}
return \%peaks;
}
sub peaks_to_gff3 {
################################################################################
# Guarda los picos en un fichero en formato gff3
# ejemplo: &peaks_to_gff3(<ref_peaks>, <path_to_gff>)
################################################################################
print STDERR "\nPrinting peaks in GFF3\n";
my $ref_peaks = shift;
my %peaks = %{$ref_peaks};
my $gff = shift;
open OUT, ">$gff" or die $!;
foreach my $key (keys %peaks) {
print OUT "$peaks{$key}{chrom}\tAnABlast\tPeak\t$peaks{$key}{start}\t$peaks{$key}{end}\t$peaks{$key}{top}\t$peaks{$key}{strand}\t.\tID=$key;frame=$peaks{$key}{frame};stop_codons=$peaks{$key}{stop_codon};amino_acids=<textarea readonly=\"\" rows=\"7\" cols=\"62\" class=\"fasta\">$peaks{$key}{aa}</textarea></div>,\n";
}
close OUT or die $!;
}
sub peaks_to_fasta {
################################################################################
# Guarda la secuencia de aa de los picos en un fichero en formato fasta
# ejemplo: &peaks_to_fasta(<ref_peaks>, <path_to_fasta>)
################################################################################
print STDERR "\nPrinting peak seqs in FASTA\n";
my $ref_peaks = shift;
my %peaks = %{$ref_peaks};
my $fasta = shift;
open OUT, ">$fasta" or die $!;
foreach my $key (keys %peaks) {
print OUT ">$key $peaks{$key}{chrom}:$peaks{$key}{start}..$peaks{$key}{end}\n";
print OUT "$peaks{$key}{aa}\n";
}
close OUT or die $!;
}
sub peaks_elong_orf_search {
################################################################################
# Busca ORF en los picos
# ejemplo: &orf_search(<ref_peaks>, <sequence_reference>)
################################################################################
my $ref_peaks = shift;
my %peaks = %{$ref_peaks};
my $seq_ref = shift;
my %seq = %{$seq_ref};
print STDERR "\nSearching ORF in peaks\n";
my %orf;
my $c;
foreach my $key (keys %peaks) {
my $sub_frame = &frame_calc($peaks{$key}{frame}, $peaks{$key}{start}, $peaks{$key}{end}, $seq{$peaks{$key}{chrom}}{length});
my $nn;
my $start;
my $end;
if ($peaks{$key}{frame} > 0) {
$start = $peaks{$key}{start} + $sub_frame -1;
my $rest = ($peaks{$key}{end} - $start + 1) % 3;
$end = $peaks{$key}{end} - $rest;
while (1) {
if ($start <= 1) {
$start = $peaks{$key}{frame};
last;
}
my $codon = substr ($seq{$peaks{$key}{chrom}}{nn}, $start -1, 3);
if ($codon eq "TGA" or $codon eq "TAA" or $codon eq "TAG" or $codon eq "NNN") {
last;
}
else {
$start -= 3;
}
}
while (1) {
if ($end >= $seq{$peaks{$key}{chrom}}{length} - 3) {
$end = $seq{$peaks{$key}{chrom}}{length};
last
}
my $codon = substr ($seq{$peaks{$key}{chrom}}{nn}, $end, 3);
if ($codon eq "TGA" or $codon eq "TAA" or $codon eq "TAG" or $codon eq "NNN") {
$end += 3;
last;
}
else {
$end += 3;
}
}
$nn = substr ($seq{$peaks{$key}{chrom}}{nn}, $start -1, $end - $start +1);
my $f = 0;
for (my $i = 0; $i <= length($nn); $i += 3) {
my $codon = substr($nn, $i, 3);
if ($codon eq "ATG" or $codon eq "ATA" or $codon eq "ATC" or $codon eq "GTG" or $codon eq "TTG" or $codon eq "CTG" or $codon eq "ATT") {
if ($f == 0) {
$c++;
$f = 1;
$orf{"$key"."_ORF_$c"}{start} = $start + $i;
$orf{"$key"."_ORF_$c"}{parent} = $key;
$orf{"$key"."_ORF_$c"}{chrom} = $peaks{$key}{chrom};
$orf{"$key"."_ORF_$c"}{strand} = $peaks{$key}{strand};
$orf{"$key"."_ORF_$c"}{frame} = $peaks{$key}{frame};
}
}
elsif ($codon eq "TGA" or $codon eq "TAA" or $codon eq "TAG") {
if ($f == 1) {
$f = 0;
$orf{"$key"."_ORF_$c"}{end} = $start + $i +2;
$orf{"$key"."_ORF_$c"}{nn} = substr ($seq{$peaks{$key}{chrom}}{nn}, $orf{"$key"."_ORF_$c"}{start} -1, $orf{"$key"."_ORF_$c"}{end} - $orf{"$key"."_ORF_$c"}{start} + 1);
$orf{"$key"."_ORF_$c"}{aa} = &dna2aa($orf{"$key"."_ORF_$c"}{nn}, 1);
delete $orf{"$key"."_ORF_$c"} if (length $orf{"$key"."_ORF_$c"}{aa} <= 5);
}
}
}
}
else {
$end = $peaks{$key}{end} - $sub_frame + 1;
my $rest = ($end - $peaks{$key}{start} + 1) % 3;
$start = $peaks{$key}{start} + $rest;
while (1) {
if ($start <= 1) {
$start = 1;
last;
}
my $codon = substr ($seq{$peaks{$key}{chrom}}{nn}, $start -1, 3);
my $rcodon = &reverse_comp($codon);
if ($rcodon eq "TGA" or $rcodon eq "TAA" or $rcodon eq "TAG" or $rcodon eq "NNN") {
last;
}
else {
$start -= 3;
}
}
while (1) {
if ($end >= $seq{$peaks{$key}{chrom}}{length} - 3) {
$end = $seq{$peaks{$key}{chrom}}{length} + $peaks{$key}{frame} + 1;
last;
}
my $codon = substr ($seq{$peaks{$key}{chrom}}{nn}, $end, 3);
my $rcodon = &reverse_comp($codon);
if ($rcodon eq "TGA" or $rcodon eq "TAA" or $rcodon eq "TAG" or $rcodon eq "NNN") {
$end += 3;
last;
}
else {
$end += 3;
}
}
$nn = substr ($seq{$peaks{$key}{chrom}}{nn}, $start -1, $end - $start +1);
#print "\n$peaks{$key}{chrom}:$start..$end\t$peaks{$key}{frame}\n$nn\n";
$nn = &reverse_comp($nn);
my $f = 0;
for (my $i = 0; $i <= length($nn); $i += 3) {
my $codon = substr($nn, $i, 3);
if ($codon eq "ATG" or $codon eq "ATA" or $codon eq "ATC" or $codon eq "GTG" or $codon eq "TTG" or $codon eq "CTG" or $codon eq "ATT") {
if ($f == 0) {
$c++;
$f = 1;
$orf{"$key"."_ORF_$c"}{end} = $end - $i;
$orf{"$key"."_ORF_$c"}{parent} = $key;
$orf{"$key"."_ORF_$c"}{chrom} = $peaks{$key}{chrom};
$orf{"$key"."_ORF_$c"}{strand} = $peaks{$key}{strand};
$orf{"$key"."_ORF_$c"}{frame} = $peaks{$key}{frame};
}
}
elsif ($codon eq "TGA" or $codon eq "TAA" or $codon eq "TAG") {
if ($f == 1) {
$f = 0;
$orf{"$key"."_ORF_$c"}{start} = $end - $i - 2;
$orf{"$key"."_ORF_$c"}{nn} = &reverse_comp(substr ($seq{$peaks{$key}{chrom}}{nn}, $orf{"$key"."_ORF_$c"}{start} -1, $orf{"$key"."_ORF_$c"}{end} - $orf{"$key"."_ORF_$c"}{start} +1));
$orf{"$key"."_ORF_$c"}{aa} = &dna2aa($orf{"$key"."_ORF_$c"}{nn}, 1);
delete $orf{"$key"."_ORF_$c"} if (length $orf{"$key"."_ORF_$c"}{aa} <= 5);
}
}
}
}
}
foreach my $key (keys %orf) {
if (!$orf{$key}{start} or !$orf{$key}{end}) {
delete $orf{$key};
}
}
return \%orf;
}
sub new_orf_search {
################################################################################
# Busca ORF en los picos con un núevo método
# ejemplo: &orf_search(<ref_peaks>, <sequence_reference>)
################################################################################
my $ref_peaks = shift;
my %peaks = %{$ref_peaks};
my $seq_ref = shift;
my %seq = %{$seq_ref};
print STDERR "\nSearching ORF in peaks\n";
my %orf;
my $c;
foreach my $key (keys %peaks) {
my $nn; # Secuencia de nucleótidos extraida
my $frame_start; # posición de inicio del frame
my $rest; # resto de la posición de inicio cuando lo dividimos entre 3
my $objetiv_rest; # resto que quiero tener para trabajar con el frame correcto
my $frame_end; # posición final del frame
if ($peaks{$key}{frame} > 0) { # Para los frames positivos
# cálculo de la posición de inicio del frame
if ($peaks{$key}{frame} == 1) {
$objetiv_rest = 0;
}
elsif ($peaks{$key}{frame} == 2) {
$objetiv_rest = 1;
}
elsif ($peaks{$key}{frame} == 3) {
$objetiv_rest = 3;
}
$frame_start = $peaks{$key}{start}; # iniciamos el frame en la posición de inicio del pico
$rest = $peaks{$key}{start} % 3;
for (1..6) { # Corregimos el inicio hasta tener el resto adecuado
if ($rest != $objetiv_rest) {
$frame_start++;
}
elsif ($rest == $objetiv_rest) {
last;
}
}
$frame_end = $frame_start + 3; # iniciamos el final del frame en el siguiente codon al inicio del frame
# búsqueda del primer codon de stop previo al inicio del frame
while (1) {
if ($frame_start <= 1) {
$frame_start = $peaks{$key}{frame};
last;
}
my $codon = substr ($seq{$peaks{$key}{chrom}}{nn}, $frame_start -1, 3);
if ($codon eq "TGA" or $codon eq "TAA" or $codon eq "TAG" or $codon eq "NNN") {
last;
}
else {
$frame_start -= 3;
}
}
# Búsqueda del siguiente codon de stop
while (1) {
if ($frame_end >= $seq{$peaks{$key}{chrom}}{length} - 3) {
$frame_end = $seq{$peaks{$key}{chrom}}{length};
last;
}
my $codon = substr ($seq{$peaks{$key}{chrom}}{nn}, $frame_end -1, 3);
if ($codon eq "TGA" or $codon eq "TAA" or $codon eq "TAG" or $codon eq "NNN") {
$frame_end += 3;
if ($frame_end < $peaks{$key}{end}) { # Si encuentra un codon de stop comprueba que ha superado el final del pico
next;
}
else {
last;
}
}
else {
$frame_end += 3;
}
}
$nn = substr ($seq{$peaks{$key}{chrom}}{nn}, $frame_start -1, $frame_end - $frame_start +1); # Extrae la secuencia de nucleótidos
my $f = 0;
for (my $i = 0; $i <= length($nn); $i += 3) {
my $codon = substr($nn, $i, 3);
if ($codon eq "ATG" or $codon eq "ATA" or $codon eq "ATC" or $codon eq "GTG" or $codon eq "TTG" or $codon eq "CTG" or $codon eq "ATT") {
if ($f == 0) {
$c++;
$f = 1;
$orf{"$key"."_ORF_$c"}{start} = $frame_start + $i;
$orf{"$key"."_ORF_$c"}{parent} = $key;
$orf{"$key"."_ORF_$c"}{chrom} = $peaks{$key}{chrom};
$orf{"$key"."_ORF_$c"}{strand} = $peaks{$key}{strand};
$orf{"$key"."_ORF_$c"}{frame} = $peaks{$key}{frame};
}
}
elsif ($codon eq "TGA" or $codon eq "TAA" or $codon eq "TAG") {
if ($f == 1) {
$f = 0;
$orf{"$key"."_ORF_$c"}{end} = $frame_start + $i +2;
$orf{"$key"."_ORF_$c"}{nn} = substr ($seq{$peaks{$key}{chrom}}{nn}, $orf{"$key"."_ORF_$c"}{start} -1, $orf{"$key"."_ORF_$c"}{end} - $orf{"$key"."_ORF_$c"}{start} + 1);
$orf{"$key"."_ORF_$c"}{aa} = &dna2aa($orf{"$key"."_ORF_$c"}{nn}, 1);
delete $orf{"$key"."_ORF_$c"} if (length $orf{"$key"."_ORF_$c"}{aa} <= 5);
}
}
}
}
else { # para los frames negativos
# cálculo de la posición de inicio del frame
if ($peaks{$key}{frame} == -1) {
$objetiv_rest = 0;
}
elsif ($peaks{$key}{frame} == -2) {
$objetiv_rest = 1;
}
elsif ($peaks{$key}{frame} == -3) {
$objetiv_rest = 3;
}
$frame_start = $peaks{$key}{end};
$rest = ($seq{$peaks{$key}{chrom}}{length} - $frame_start) % 3;
for (1..6) { # Corregimos el inicio hasta tener el resto adecuado
if ($rest != $objetiv_rest) {
$frame_start--;
}
elsif ($rest == $objetiv_rest) {
last;
}
}
$frame_end = $frame_start - 3; # Iniciamos el final del frame en el codon anterior
# Buscamos el primer codon de fin despues de la posición de inicio
while (1) {
if ($frame_start >= $seq{$peaks{$key}{chrom}}{length} - 3) {
$frame_start = $seq{$peaks{$key}{chrom}}{length} + $peaks{$key}{frame};
last;