Hello everyone! I'm trying to get intergenic sequences for many genebank files. To do so I'm trying something like this:

use strict;
use warnings;
use autodie;
use Data::Dump;
use Data::Dumper;
use Bio::SeqIO;
STDOUT->autoflush(1);

my $gbffpath='C:\My gbff path';

opendir(DIR, "$gbffpath");
my @files= grep {/\.gbff$/ } readdir(DIR);
closedir(DIR);
my $featc=0;

foreach my $gbfffile (@files){
    my $in = Bio::SeqIO->new(-file=>"<$gbffpath\\$gbfffile", -format=>
+"genbank");
    my @allpos;
    my $obj = $in->next_seq();
    my @features = $obj->get_SeqFeatures();
    my @two = shift @features;
    foreach my $fefe  (@features){
        my $pt = $fefe->primary_tag();
        if ($pt eq "CDS" or $pt eq "rRNA" or $pt eq "tRNA") {
            $featc++;
            last if $featc==4;
            push @two, $fefe;
            my $starti= $two[1]->start();
            my $endi; my $end; my $start;
            if($two[0]->primary_tag() eq "source"){
                $endi = $starti;
                $end = $endi +1;
                $start = $starti -1;
                shift @two;
            }
            else{
                $endi = $two[0]->end();
                $end = $endi +1;
                $start = $starti -1;
                #do something else
                # change $start to be -21 nucleotides
                # my $subseq = $obj->subseq($end,$start) unless ($end>
+=$start);
                my $subseq = $obj->subseq($end,$start) unless ($end>=$
+start);
                shift @two;

                dd ($gbfffile,$pt,"\$two\[0\]:","endi:$endi","\$two\[1
+\]:","starti:$starti","after:","end:$end","start:$start",$subseq);
            }
    }
}

[download]

However, $subseq always returns undefined and I cannot understand why. If I change that to $obj->seq() the same happens. Can someone please help me?

Edit: this question was cross-posted at https://www.biostars.org/p/152157/.

In reply to Trying to get intergenic sequences using BioPerl by Sosi

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