Add this to your code:

use Data::Dumper; $Data::Dumper::Sortkeys=1; print Dumper(\%third);
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Does this output look right to you?:

$VAR1 = {
          'AC' => 0,
          'AT' => 0,
          'C' => 4,
          'TC' => 0
        };
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If not, I agree that Line 15 is not what you want. See also Basic debugging checklist. How about:

    $third{  substr($tri,0,1) . substr($tri,2,1) }++; # Line 15
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