To find the sequence alignment program that performs best under all non-trivial situations (e. g. reference APSI ≤ 80 %), we did a comparison of all those programs previously identified [22] to be top ranking. If available we used the newest program versions and optimized parameters. In the comparison we included the RNA version of PROBCONS [33] (PROBCONSRNA; see [34]) whose parameters have been estimated via training on the BRAliBase II datasets. We applied Friedman rank sum tests to each alignment set with a fixed number of sequences. Results are summarized in Table 5. MAFFT version 5 [25] with the option "G-INS-i" ranks first throughout all test-sets. This option is suitable for sequences of similar lengths, recommended for up to 200 sequences, and uses an iterative (COFFEE-like [35]) refinement method incorporating global pairwise alignment information. This option clearly outperforms the default option "FFT-NS-2", which uses only a progressive method for alignment. MUSCLE and PROBCONSRNA rank second and third place.