Comparison of three methods for generating 20 controls from each input sequence pair. ROC curves comparing three methods for generating a set of 20 controls from an input sequence pair to determine the z score for ncRNA classification using the Dynalign-computed ΔG°total. The test set contains 755 5S rRNA and 896 tRNA sequence pairs, plus one negative sequence pair generated from each real sequence pair, yielding 3,302 trial pairs total. All tests are run with the parameter M = 8. "dinuc controls" (green): controls are generated by sampling from a first-order Markov chain, approximately preserving dinucleotide frequencies of each original sequence. "AE controls" (orange): controls are generated by the Altschul-Erikson dinucleotide shuffle, exactly preserving dinucleotide frequencies of each original sequence. "column controls" (blue): controls are generated by a columnwise shuffle of a global sequence alignment, without regard for gap placement or local conservation.