For statistical analyses we computed the BRALISCORE for each alignment. To rate the alignment programs or program options, we ranked these scores after averaging over all datasets. Because the score distributions cannot be assumed to be either normal or symmetric, we used as non-parametric tests the Friedman rank sum and the Wilcoxon signed rank test. R's Friedman test was accommodated to calculate the ranking. Afterwards the Wilcoxon test determined which programs or options pairwisely differ significantly. As already shown in [22] programs generally perform equally well above sequence similarity of about 80 %; that is, with such a similarity level the alignment problem becomes almost trivial. To avoid introduction of a bias due to the large number of high-homology alignments with a reference APSI > 80 %, we only used alignments with a reference APSI ≤ 80 % for the statistical analyses.