A total of 16 pseudoknotted reference structures from different RNA types were collected, which have strong experimental support. For each reference structure, a supporting set of 10 evolutionarily related sequences was obtained from the RFAM database (Gardner et al., 2010) . Note that for the vast majority of supporting sequences, no experimentally determined structures are available. The average pairwise sequence identities vary from 55% to 99%. Given a reference structure, the performance of prediction algorithms is evaluated in terms of sensitivity (S), i.e. the percentage of base pairs in the reference structure, which are predicted correctly, as well as positive predictive value (PPV), i.e. the percentage of predicted pairs, which are in the reference structure. The Matthews correlation coefficient (MCC) is also reported and is in the range from À1 to 1, where 1 corresponds to a perfect prediction and À1 to a prediction that is in total disagreement with the reference structure. The performance of each method for predicting the reference structure was evaluated as described in Gardner and Giegerich (2004) .