# my %freq; #<---- uncomment this somewhere before your loop  
#                  through the file to instantiate the hash

     if ($line=~m/$sequence(.{11})(.{11})$sequence/){
        $freq{$1}++;
        $freq{$2}++;
     }
}
# after all lines are read in....

# sort through the sequences, highest frequency to lowest
for my $seq (sort { $freq{$b} <=> $freq{$a} } keys %freq){
    print "$seq    $freq{$seq}\n";
}