False negatives due to missing sequence data. (A) A branch of the 16S Firmicutes tree containing Geobacillus subterraneus LTs, shaded blue (indicating no evidence for a stem) by both methods. (B) The RNAClust consensus structure, where no LT stem is observed. Labels T1 and T2 denote trailer–trailer helices, and L denotes a leader–leader helix. (C) The RNAClust consensus structure with an adjusted trailer length of 500 nts, where an LT stem is observed. The same numbering schema denotes helices between (B) and (C). The increased trailer length captures the LT stem, indicating that an LT stem exists in G. subterraneus, and that both the shuffling method (for which even fewer trailer nucleotides are gathered due to a downstream gene annotation) and the covariation method under default settings simply do not gather enough trailer nucleotides for this species. Full-page figures for each structure in (B) and (C) are available in Supplemental Material D.
