Error with Bio::Seq Translate

[dmbNEWB]

Hi all! I am new to perl, and this is my first post ever!

I am trying to translate nucleic acid sequences into amino acid sequences. I create a script (below). When I run the script with only the translate frames 0 & 1, then the script runs fine. Upon adding in the translate frame 2, the script stops working and I receive errors (below). Also, I tried running the translate frame 2 by itself, but receive the same errors.

I've tried searching these errors/message individually to troubleshoot myself, but I can't figure it out. Any guidance is much appreciated!

Script:

#!bin/perl -w
use strict;
use warnings;
use Bio::SeqIO;


#script to translate nucleotide sequences to amino acid sequences in f
+irst three frames

my $in = Bio::SeqIO->new(-file => "sequence.fasta", -format => "fasta"
+);
my $out = Bio::SeqIO->new(-file => ">Frame1.fasta", -format => "fasta"
+);
my $out2 = Bio::SeqIO->new(-file => ">Frame2.fasta", -format => "fasta
+");
my $out3 = Bio::SeqIO->new(-file => ">Frame3.fasta", -format => "fasta
+");

while ( my $seq = $in->next_seq() ) {
        my $prot = $seq->translate();
        $out->write_seq($prot);
        my $prot2 = $seq->translate(-frame => 1);
        $out2->write_seq($prot2);
        my $prot3 = $seq->translate(-frame => 2);
        $out3->write_seq($prot3);
        }

Errors: substr outside of string at /sw/lib/perl5/5.16.2/Bio/PrimarySeqI.pm line 644, <GEN0> line 5013. ------------- EXCEPTION: Bio::Root::Exception ------------- MSG: Calling translate without a seq argument! STACK: Error::throw STACK: Bio::Root::Root::throw /sw/lib/perl5/5.16.2/Bio/Root/Root.pm:472 STACK: Bio::Tools::CodonTable::translate /sw/lib/perl5/5.16.2/Bio/Tools/CodonTable.pm:411 STACK: Bio::PrimarySeqI::translate /sw/lib/perl5/5.16.2/Bio/PrimarySeqI.pm:648 STACK: translateframe.pl:11 -----------------------------------------------------------

[PerlSufi]

I don't really use the Bio::Seq* namespace much, so I am not completely sure- but maybe the problem is with this line:

my $prot = $seq->translate();

..not giving it a 'seq' argument.
UPDATE:
It looks like you have to give translate() a Bio::Seq object, not a Bio::SeqIO object.
Sample input and output would also help ;)

[dmbNEWB]

Thanks for your reply! Well, it's only the  my $prot3 = $seq->translate(2); part that does not work, which makes me think the input is fine since it works for the $prot and $prot2.

However, here is sample input if it helps! The file contains 194,503 of these sequences, but here are a couple for an idea:

>3HSAA000001 CA000001
tgcagccgcgggcccagggtgctgttggtgtcctcagaagtgccggggattctggactgg
ctccctcccctcctgttgcagcacaaggccggggtctctggggggctggagaagcctccc
tcattcctcccaggaattaataaatgtgaagagaggctctgtttaaaatgtctttggact
cccagggctgagtgggctgggatctcgtgtcctcaa
>3HSAA000002 CA000002
tgcagccgcgggcccagggtgctgttggtgtcctcagaagtgccggggattctggactgg
ctccctcccctcctgttgcagcacaaggccggggtctctggggggctggagaagcctccc
tcattcctcccaggaattaataaatgtgaagagaggctctgtttaaaatgtctttggact
cccagggctgagtgggctgggatctcgtgtcctcaaagtggatgggttctggggtggctc
ctgaggtagaggagtggagaactggctcttaagagcacagttttcttttcttttttcttt
tttgagacagggtctcacactgtcacccaggctggagtgcagtgttgcagtccggctcac
tgcagttttgacctcccaggctcaagcgatcctcctgcctcagcctcccaagtagctggg
agcctgggcatgcatcgccacgtctggctaattattattttttgtagacagggtctcact
gtgttgcccaagcttgtcttgaactcctggccttaagtgatcctcccacctcagcctcct
gagtagctgggactacaggcatgagccaccatgcctggccaactcacatttttctttcta
ttatttattttttgtagagatgagtctcactatgttgcccaggctggtcttgaactcttg
ggctcaagtgatcctcccgcctcagcctcccaaagtgttgggattacaggtgtgagccac
agcacctggccaaccaacacttctcagggcctctttcatctgtgctcttccaggatgctg
cctcttactccctgggcacctcggcctggtcccagcaggtatgggcagttgcttgaggct
ccagacatactcacctctacctcgaccacatcaaccccatcaccaagaggaggttcaggg
aagctgcattttgtggtcttgtcctcccagtccagggtggtagtgctgggctgttcccag
cctcccacagcagagctggacgctgtggaaatggctggattcctctgtgttctttcccat
tatccctcagctctgagtcctcttgtgccatctgacatctgatgccttcccaaccactgc
cgagtttctgcctggagcagggcctcaaggccctggcacacagaagatgcgtatcagtat
tatcaaccaatagttgatgaattgtgtttttcaacgaatttgctagtgatctggtttact
gccttagtaatatctagttcctaatatgcctatgccttttaatgtctgcacagtctatga
tgatgtcacttctctcaagcctgatgtgtcctctctctctttcttgctagtggtttatca
atttttaaaaccttttcttgtttatttgtttttgagacagaattttgctcttgttgccca
ggctggagtgcaatggcgtgatcttggctcacagcaacctctacctcctgggttcaagtg
attctcgtgcctcagcctcctgagtagctgggctgacaggcacccaccaccacacccagc
taatttttatgtttttagtagagatggggtttcaccttgttgggtggccaggctggtctc
caactcctgagctcaagtgatccacccgcctcagcctcccaaagtgctgggattacaggg
gtgagccaccacgcccagccccagcttagttttttaaaaagtttattttagccattctaa
taggtatgtagacatatctaatagtggttttccttgcgtttccttaatgtctgatgatgt
taagcattttttccccaagtgcttagttgccatctatatagcatctttgatgaaatgtct
gtttatatattttgcacactttaaaatattgggttgttttcttt
>3HSAA000003 CA000003
tgcagccgcgggcccagggtgctgttggtgtcctcagaagtgccggggattctggactgg
ctccctcccctcctgttgcagcacaaggccggggtctctggggggctggagaagcctccc
tcattcctcccaggaattaataaatgtgaagagaggctctgtttaaaatgtc

[PerlSufi]

You're welcome. And what is the goal of the script? To convert the DNA to ribose nucleic acid (RNA) ?
UPDATED CODE TO REFLECT TRANSLATING TO RNA
For starters, here is code I made (with acknowledgements to the Perl Monks for help) to parse a fasta file:

use strict;
use warnings;
use Data::Dumper;

my $file = '/path/to/your/file/here.fasta';
open (my $fh, '<', $file) or die "Could not open file '$file' $!";

my (%sequence_hash, $header);

while ( my $line = <$fh> ) {
    chomp($line);
    if ( $line =~ m/^>(.*)/ ) {
        $header = $1;
    }
    else {
        $sequence_hash{$header} .= $line;
    }
}

# look at the header and sequence
print Dumper(\%sequence_hash);

my @translated_sequences;

foreach my $seq ( keys %sequence_hash ) {
      $sequence_hash{$seq} =~ s/t/u/ig;
      push @translated_sequences, $sequence_hash{$seq};
}

print Dumper(\@translated_sequences);

[GotToBTru]

In the docs, there are no parentheses after the method call, so maybe try

my $prot = $seq->translate;

Strictly a wild guess.

[dmbNEWB]

Thanks for your response, but the parentheses are necessary to designate the translation frame.