use warnings; use strict;

open GENE, '<', '\test.txt' or die("Unable to read: $!");
my @rolling = (undef, undef, undef);
my %count;
my $cnt;

until (eof GENE) {
   my $char;
   read(GENE,$char,1);
   next unless $char=~/[AGCT]/; #make sure it's a valid char;

   shift @rolling;
   push @rolling, $char;

   next unless defined $rolling[2];
   $count{$rolling[2]}++;            #one-char count

   next unless defined $rolling[1];
   $count{join('',@rolling[1,2])}++; #two-char count

   next unless defined $rolling[0]; 
   $count{join('',@rolling)}++;      #three-char count
}