Running the while loop 
while( my $seq = $in->next_seq  ) {
      print $i++, " : ", $seq->seq(), "\n";
   }

[download]
will read until the end of the filehandle. (That is why the loop ended in the first place). So subsequently calling next_seq on the $in object will give you the error mesg you are seeing. You can
  1. open SeqIO object outside of trial loop, and put seek(INFILE,0) inside the loop. Note that if the SeqIO object gets destroyed (or goes out of scope), you will need to add the flag -noclose => 1 option when initing the SeqIO object or else the filehandle is closed. But if you are going to do this, just move the initialization of the SeqIO object outside the loop.
  2. re-open the file each time in the list (move the Bio::SeqIO initialization into the trial loop)
  3. Or read all the sequences in at once and keep them in memory. (put them into an array). 
    my @seqs; 
while(my $s = $in->next_seq ) { push @seqs, $s; }
# now do your loop of trials

    [download]
#3 might not work depending on how many sequences and how big they are.

In reply to Re^3: How to avoid using array in concatenating string of multiple lines by stajich
in thread How to avoid using array in concatenating string of multiple lines OR How To Read FASTA by monkfan

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