Hi tilly, Thanks so much for your advice. I set up the tie earlier in the program than the database structure creation:

use Bio::AlignIO;
use Bio::SeqIO;
use Fcntl qw(O_RDWR O_CREAT);
use DBM::Deep;
use Data::Dumper;

<br>
...<br>
my $db = tie %positions_hash, "DBM::Deep", "positions.db";
<br>...<br>
# hash creation
[download]

This appears to work, but the only drawback is slowness. Guess I can't get away from that by only tie-ing after hash creation because that produces the error:
DBM::Deep: Cannot sysopen file 'positions.db': No such file or directory.
Thanks again.