G'day azheid,

Cutting your code down to a skeleton where I think your two biggest problems are:

if(!($permute)){
    open(SEQ,'<',$sequence_fname)||die "Cannot open sequence file\n";
    while (my @line=split (/\t/,<SEQ>)){
        ...
    }   
    ...
{   
else{
    for (...) {
        ...
        open(SEQ,'<',$sequence_fname)||die "Cannot open sequence file\
+n";#open the sequence file
        while (my @line=split (/\t/,<SEQ>)){#for each sequence record 
+the nmer information
            ...
        }
        close SEQ;
        open(OUT,">>$out")||die "Cannot open $out\n";
        foreach my $key(keys %ktc){
            ...
            print OUT ...
            ...
        }
        close OUT;
    }
}
[download]

You open the SEQ file, read the data from disk, and parse it multiple times: you only need to do this once.

Also, you open and close the OUT file for appending multiple times: you only need to do this once.

Without making changes to your coding style, I suspect this, which does only open those files once, would be substantially faster:

my @seq_data;
open(SEQ,'<',$sequence_fname)||die "Cannot open sequence file\n";
while (<SEQ>) {
    push @seq_data, [split /\t/];
}
close SEQ;

if(!($permute)){
    for (@seq_data) {
        my @line = @$_;
        ...
    }
    ...
{
else{
    open(OUT,">>$out")||die "Cannot open $out\n";

    for (...) {
        ...
        for (@seq_data) {
            my @line = @$_;
            ...
        }

        foreach my $key(keys %ktc){
            ...
            print OUT ...
            ...
        }
    }

    close OUT;
}
[download]

There may be other areas where substantial gains could be made, but I haven't looked beyond the two I/O ones at this point.

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