I'd think you didn't get your reversal of the tag-sequence right

I modified this to:

         print "tag \t= $tag\n";
         my $RC_tag = reverse($tag);
         print "RC_tag \t= $RC_tag\n";
         $RC_tag =~ tr/ACTGactg/TGACtgac/;
         print "RC_tag \t= $RC_tag\n";
[download]

This would print:

 tag         = ACGACACGTAT
 RC_tag      = TATGCACAGCA
 RC_tag      = ATACGTGTCGT
[download]

Which one would be correct?

In your original code,

        my $tag = $tag_obj->seq;
        print "tag = ",$tag,"\n";
        my $RC_tag = reverse $tag =~ tr/ACTGactg/TGACtgac/;
[download]

$RC_tag would be the number '11', which probably isn't what you want.

Regards

mwa