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#!/usr/local/bin/perl
package main;
our $SEE;
package Longest_orf;
use strict;
use warnings;
use Nuc_translator;
use Carp;
## if allow_partials is set, partial orfs are included in the analysis.
# below used to be static, now instance vars.
#my $ALLOW_5PRIME_PARTIALS = 0; #allow for lacking start codon in logest orf.
#my $ALLOW_3PRIME_PARTIALS = 0; #allow for lacking stop codon in longest orf.
#my $FORWARD_STRAND = 1; #default set to true (analyze forward strand)
#my $REVERSE_STRAND = 1; #default set to true.
#my $ALLOW_NON_MET_STARTS = 0; #allow for non-methionine start codons.
sub new {
shift;
## This object stores the longest ORF identified.
my @stop_codons = &Nuc_translator::get_stop_codons(); # live call, depends on current genetic code.
print "Stop codons in use: @stop_codons, set dynamically via current Nuc_translator settings.\n" if $SEE;
unless (@stop_codons) {
confess "Fatal, no stop codons set";
}
my $obj = { pep_seq => undef,
nt_seq => undef,
length => undef, #length of nt_seq
end5 => undef,
end3 => undef,
all_ORFS=>[], #container holds all ORFs found in order of decreasing length. Use orfs() method to retrieve them.
stop_codons => [@stop_codons],
## ORF settings
ALLOW_5PRIME_PARTIALS => 0,
ALLOW_3PRIME_PARTIALS => 0,
FORWARD_STRAND => 1,
REVERSE_STRAND => 1,
ALLOW_NON_MET_STARTS => 0
};
bless ($obj);
return ($obj);
}
## can include partial orfs at end of sequence.
sub allow_partials {
my $self = shift;
die unless (ref $self);
$self->{ALLOW_5PRIME_PARTIALS} = 1;
$self->{ALLOW_3PRIME_PARTIALS} = 1;
if ($SEE) {
print "Longest_orf: allowing both 5' and 3' partials.\n";
}
}
sub allow_5prime_partials {
my $self = shift;
die unless (ref $self);
$self->{ALLOW_5PRIME_PARTIALS} = 1;
if ($SEE) {
print "Longest_orf: allowing 5prime partials.\n";
}
}
sub allow_3prime_partials {
my $self = shift;
die unless (ref $self);
$self->{ALLOW_3PRIME_PARTIALS} = 1;
if ($SEE) {
print "Longest_orf: allowing 3prime partials\n";
}
}
sub forward_strand_only {
my $self = shift;
die unless (ref $self);
$self->{REVERSE_STRAND} = 0;
if ($SEE) {
print "Longest_orf: forward strand only.\n";
}
}
sub reverse_strand_only {
my $self = shift;
die unless (ref $self);
$self->{FORWARD_STRAND} = 0;
if ($SEE) {
print "Longest_orf: reverse strand only.\n";
}
}
sub allow_non_met_starts {
my $self = shift;
$self->{ALLOW_NON_MET_STARTS} = 1;
if ($SEE) {
print "Longest_orf: allowing non Met start codons.\n";
}
}
sub get_longest_orf {
my $self = shift;
my $input_sequence = shift;
unless ($input_sequence) {
print STDERR "I require a cDNA nucleotide sequence as my only parameter\n";
return;
}
unless (length ($input_sequence) >= 3) {
print STDERR "Sequence must code for at least a codon. Your seq_length is too short\n";
return;
}
my @orfList = $self->capture_all_ORFs($input_sequence);
# print "Found " . scalar @orfList . " orfs.\n";
if (@orfList) {
return ($orfList[0]); # longest ORF found is first in the sorted list.
}
else {
## no ORFs found
return (undef);
}
}
sub capture_all_ORFs {
my $self = shift;
my $input_sequence = shift;
unless ($input_sequence) {
print STDERR "I require a cDNA nucleotide sequence as my only parameter\n";
return;
}
unless (length ($input_sequence) >= 3) {
print STDERR "Sequence must code for at least a codon. Your seq_length is too short\n";
return;
}
$input_sequence = lc ($input_sequence);
my (@starts, @stops, @orfs);
if ($self->{FORWARD_STRAND}) {
## analyse forward position
@stops = $self->identify_putative_stops($input_sequence);
@starts = $self->identify_putative_starts($input_sequence,\@stops);
@orfs = $self->get_orfs (\@starts, \@stops, $input_sequence, '+');
}
if ($self->{REVERSE_STRAND}) {
## reverse complement sequence and do again
$input_sequence = &revcomp ($input_sequence);
@stops = $self->identify_putative_stops($input_sequence);
@starts = $self->identify_putative_starts($input_sequence, \@stops);
push (@orfs, $self->get_orfs (\@starts, \@stops, $input_sequence, '-'));
}
if (@orfs) {
## set in order of decreasing length
@orfs = reverse sort {$a->{length} <=> $b->{length}} @orfs;
my $longest_orf = $orfs[0];
my $start = $longest_orf->{start};
my $stop = $longest_orf->{stop};
my $seq = $longest_orf->{sequence};
my $length = length($seq);
my $protein = &translate_sequence($seq, 1);
$self->{end5} = $start; ## now coord is seq_based instead of array based.
$self->{end3} = $stop;
$self->{length} = $length;
$self->{nt_seq} = $seq;
$self->{pep_seq} = $protein;
$self->{all_ORFS} = \@orfs;
}
return (@orfs);
}
sub orfs {
my $self = shift;
return (@{$self->{all_ORFS}});
}
#####################
# supporting methods
#####################
sub get_end5_end3 {
my $self = shift;
return ($self->{end5}, $self->{end3});
}
sub get_peptide_sequence {
my $self = shift;
return ($self->{pep_seq});
}
sub get_nucleotide_sequence {
my $self = shift;
return ($self->{nt_seq});
}
sub toString {
my $self = shift;
my ($end5, $end3) = $self->get_end5_end3();
my $protein = $self->get_peptide_sequence();
my $nt_seq = $self->get_nucleotide_sequence();
my $ret_string = "Coords: $end5, $end3\n"
. "Protein: $protein\n"
. "Nucleotides: $nt_seq\n";
return ($ret_string);
}
#################################
#Private methods:
sub get_orfs {
my ($self, $starts_ref, $stops_ref, $seq, $direction) = @_;
unless ($starts_ref && $stops_ref && $seq && $direction) {
confess "Error, params not appropriate";
}
my %last_delete_pos = ( 0=>-1,
1=>-1,
2=>-1); #store position of last chosen stop codon in spec reading frame.
my @orfs;
my $seq_length = length ($seq);
if ($SEE) {
print "Potential Start codons: " . join (", ", @$starts_ref) . "\n";
print "Potential Stop codons: " . join (", ", @$stops_ref) . "\n";
}
foreach my $start_pos (@{$starts_ref}) {
my $start_pos_frame = $start_pos % 3;
foreach my $stop_pos (@{$stops_ref}) {
# print "Comparing start: $start_pos to stop: $stop_pos, $direction\n";
if ( ($stop_pos > $start_pos) && #end3 > end5
( ($stop_pos - $start_pos) % 3 == 0) #must be in-frame
&& ($start_pos > $last_delete_pos{$start_pos_frame})) #only count each stop once.
{
$last_delete_pos{$start_pos_frame} = $stop_pos;
my ($start_pos_adj, $stop_pos_adj) = ( ($start_pos+1), ($stop_pos+1+2));
#print "Startposadj: $start_pos_adj\tStopPosadj: $stop_pos_adj\n";
# sequence based position rather than array-based
my ($start, $stop) = ($direction eq '+') ? ($start_pos_adj, $stop_pos_adj)
: (&revcomp_coord($start_pos_adj, $seq_length), &revcomp_coord($stop_pos_adj, $seq_length));
print "Retrieving ORF, Start: $start\tStop: $stop\n" if $SEE;
my $orfSeq = substr ($seq, $start_pos, ($stop_pos - $start_pos + 3)); #include the stop codon too.
my $protein = &translate_sequence($orfSeq, 1);
if ($protein =~ /\*.*\*/) {
confess "Fatal Error: Longest_orf: ORF returned which contains intervening stop(s): ($start-$stop, $direction\nProtein:\n$protein\nOf Nucleotide Seq:\n$seq\n";
}
my $orf = { sequence => $orfSeq,
protein => $protein,
start=>$start,
stop=>$stop,
length=>length($orfSeq),
orient=>$direction
};
push (@orfs, $orf);
last;
}
}
}
return (@orfs);
}
sub identify_putative_starts {
my ($self, $seq, $stops_aref) = @_;
my %starts;
my %stops;
foreach my $stop (@$stops_aref) {
$stops{$stop} = 1;
}
if ($self->{ALLOW_5PRIME_PARTIALS} || $self->{ALLOW_NON_MET_STARTS}) {
$starts{0} = 1 unless $stops{0};
$starts{1} = 1 unless $stops{1};
$starts{2} = 1 unless $stops{2};
}
if (! $self->{ALLOW_NON_MET_STARTS}) { #Look for ATG start codons.
my $start_pos = index ($seq, "atg");
while ($start_pos != -1) {
$starts{$start_pos} = 1;
#print "Start: $start_pos\n";
$start_pos = index ($seq, "atg", ($start_pos + 1));
}
} else {
# find all residues just subsequent to a stop codon, in-frame:
foreach my $stop (@$stops_aref) {
my $candidate_non_met_start = $stop +3;
unless ($stops{$candidate_non_met_start}) {
$starts{$candidate_non_met_start} = 1;
}
}
}
my @starts = sort {$a<=>$b} keys %starts;
return (@starts);
}
sub identify_putative_stops {
my ($self, $seq) = @_;
my %stops;
if ($self->{ALLOW_3PRIME_PARTIALS}) {
## count terminal 3 nts as possible ORF terminators.
my $seq_length = length ($seq);
$stops{$seq_length} = 1;
$seq_length--;
$stops{$seq_length} = 1;
$seq_length--;
$stops{$seq_length} = 1;
}
my @stop_codons = @{$self->{stop_codons}};
foreach my $stop_codon (@stop_codons) {
$stop_codon = lc $stop_codon;
print "Searching for stop codon: ($stop_codon).\n" if $SEE;
my $stop_pos = index ($seq, $stop_codon);
while ($stop_pos != -1) {
$stops{$stop_pos} = 1;
$stop_pos = index ($seq, $stop_codon, ($stop_pos + 1)); #include the stop codon too.
}
}
my @stops = sort {$a<=>$b} keys %stops;
return (@stops);
}
sub revcomp {
my ($seq) = @_;
my $reversed_seq = reverse ($seq);
$reversed_seq =~ tr/ACGTacgtyrkm/TGCAtgcarymk/;
return ($reversed_seq);
}
sub revcomp_coord {
my ($coord, $seq_length) = @_;
return ($seq_length - $coord + 1);
}
1;
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