Generation of RNA Structure Alignment Reference

For the construction of reference alignments we used "seed" alignments from the Rfam database version 7.0 [24 (link),23 (link)]. In most cases these alignments are hand-curated and thus of higher quality than Rfam's "full" alignments generated automatically by the INFERNAL RNA profile package [40 (link)]. Alignments with less than 50 sequences were discarded to increase the possibility for creation of subalignments (see below). The SCI (see below) for scoring of structural alignment quality is based on a combination of thermodynamic and covariation measures. Thermodynamic structure prediction becomes increasingly inaccurate with increasing sequence length – e. g. due to kinetic effects – but is widely regarded as sufficiently accurate for sequences not exceeding 300 nt in length [41 (link),42 (link)]. Thus we excluded alignments with an average sequence length above 300 nt to ensure proper thermodynamic scoring.
To each remaining seed alignment we applied a "naive" combinatorial approach that extracts sub-alignments with k ∈ {2, 3, 5, 7, 10, 15} sequences for a given average pairwise sequence identity range (APSI; a measure for sequence homology computed with ALISTAT from the squid package [43 ]). Therefore we computed identities for all sequence pairs from an alignment and selected those pairs possessing the desired APSI ± 10 %. From the remaining list of sequences we randomly picked k unique sequences. Additionally we dropped all alignments with an SCI below 0.6 to assure the structural quality of the alignments and to make sure that the SCI can be applied later to score the test alignments. This way we generated overall 18,990 reference alignments with an average SCI of 0.93; the data-set1 used in [22 (link)] consists of only 388 alignments with an average SCI of 0.89. For further details see Tables 1 and 6.