use strict;
use Bio::SeqIO;
use Bio::Tools::Run::StandAloneBlast;
my %srna;
my %genome;
my $seqio_srna = Bio::SeqIO->new(- file=>'/home/jayakuma/script/graphi
+cs/JH_TCV_lateSL.clones.filtered.23_1_2008.fa',
                 -format=>'fasta') or die "cannot open file\n";
my $seqio_large = Bio::SeqIO->new(-file => '/home/jayakuma/script/grap
+hics/TCV-jagger.fna',
                  -format =>'fasta') or die " cannot opne the file\n";

while (my $large_seq = $seqio_large->next_seq()){
    my $id = $large_seq->display_id;
    my $seq = $large_seq->seq;
    $genome{$id} = $seq;
    
    while (my $seq_srna = $seqio_srna->next_seq()){
    my $display_id = $seq_srna->display_id;
    my $seq = $seq_srna->seq;
    $srna{$display_id} = $seq;
    #rint "seq: $seq\n";
    }
    
    foreach my $seq_id (keys %srna){
    my $srnas = $srna{$seq_id};
    print "srnas: $srnas\n";
    
    foreach($srnas){
    my $blast = Bio::Tools::Run::StandAloneBlast->new(program=>'blastn
+',database=>'/home/jayakuma/script/graphics/TCV-jagger.fna');
    my $input = Bio::Seq->new(-seq=> $srnas);
    print $input->seq, "\n";
    my $blast_report = $blast->blastall($input);
    while (my $result = $blast_report->next_result()){
        my $query_length = $result->query_length();
        while (my $hit = $result->next_hit()){
        my $id = $hit->accession();
        while (my $hsp = $hit->next_hsp()){
            if($hsp->frac_identical ==1 && $hsp ->length ==$query_leng
+th){
                print "$srnas\t$id\n";
            }
        }
        }
    }
    }
}

}
[download]

srnas: TTTGCAGTATTGGACAAGCC
TTTGCAGTATTGGACAAGCC
Use of uninitialized value in pattern match (m//) at /usr/share/perl5/
+Bio/SeqIO/fasta.pm line 193, <GEN0> line 81643.
Use of uninitialized value in print at /usr/share/perl5/Bio/Root/IO.pm
+ line 407, <GEN0> line 81643.

-------------------- WARNING ---------------------
MSG: cannot find path to blastall
---------------------------------------------------
Can't call method "next_result" on an undefined value at aligning.pl l
+ine 34, <GEN0> line 81643.
[download]

May just be a typo but on line 6, "- file" should be "-file".

From the first error message, I'd guess that this was an input format error. Little things like spaces in the fasta ID could kill the parser. Check your input around line 81643. If this script works on a small portion of the input, try with a region around the error.

The second error message could just be an artifact because it really should know where "blastall" is kept. Just to make sure, check $blast->executable('blastall').

hi there I checked with both the stuffs you have mentioned. The last one the executable('blastall') didn't cause any errors. And as you have suggested shrinking the file size din't do any good, because it results in the same error but with different line numbers. I even tried with just a single seqeuence and even that results in


CAGCGATGGGGATCAAGCTC
Use of uninitialized value in pattern match (m//) at /usr/share/perl5/
+Bio/SeqIO/fasta.pm line 193, <GEN0> line 2.
Use of uninitialized value in print at /usr/share/perl5/Bio/Root/IO.pm
+ line 407, <GEN0> line 2.

-------------------- WARNING ---------------------
MSG: cannot find path to blastall
---------------------------------------------------
[download]

I have also included a bit of my input sequence:

>EH0MKSX01A0000.1
CAGCGATGGGGATCAAGCTC
>EH0MKSX01A006U.1
ATTTGATAAAGCCATCGGAGGCTT
>EH0MKSX01A00BH.1
CAGCCGAGGGACCCACGATAC
>EH0MKSX01A00O3.1
CCAGCACGTATCCGTGGACGC
>EH0MKSX01A00VX.1
CGGATAGCGGGGCGGATATAGAT
>EH0MKSX01A0133.1
AAGATGCGTCGAACCTTCGGGG
>EH0MKSX01A01AH.1
GCGTATGAGGAGCCATGCAT
>EH0MKSX01A01AK.1
AATACAAGAGTAGCTAAGTTGTCC
>EH0MKSX01A01FV.1
CGATCCAATGATGCAGCCTT
>EH0MKSX01A01IQ.1
CAGGAGGAGAGTTCGTCAAA
>EH0MKSX01A01M9.1
AAACAGACCGCCCGCGCAGCG
>EH0MKSX01A01MX.1
ATGGTGAAGGGTGGGTCATGGT
>EH0MKSX01A01PQ.1
ATGATGCCGCCCAACTCGGTGA
>EH0MKSX01A01SX.1
CCCATGACTGGGAGGTCGTGGT
>EH0MKSX01A01WT.1
AGGGGCGGTTTGCCATGCATGC
[download]

Sorry if thats to silly but I couldn't spot it.. getting nuts of this:(
any help please, thanks,