The software flags sequences that have uncertain assignments or in which no HMM regions could be detected in either orientation, suggesting the presence of sequence anomalies. We evaluated the reliability of the software by screening all bacterial (387 520) and archaeal (19 261) 16S sequences deposited in the SILVA database release 102 (Fig. 1a); mitochondrial and chloroplast sequences were excluded beforehand. Because the SILVA database stores all entries in a well-curated

multiple sequence find protocol alignment, all these entries should be present in the 5′–3′ orientation. On a 3 GHz dual-core computer, v-revcomp processed the bacterial and archaeal datasets in 252 and 8 min, respectively. All sequences except one bacterial entry were assigned as being in the 5′–3′ orientation, representing a detection accuracy of virtually 100%. The software Selleck GSK269962 flagged 40 (0.01%) sequences that showed the detection of either one HMM (37 cases), two HMMs (two cases) or three HMMs (one case) in the reverse complementary orientation; however, the majority of HMMs (i.e. 9–16) were detected in the input orientation. We studied these 40 uncertain sequences in more detail using blast against

NCBI GenBank (Benson et al., 2010) as well as through pairwise sequence alignments against an Escherichia coli reference rRNA operon (GenBank accession J01695, Prestle et al., 1992) where necessary. Fifteen cases (37.5%) were reverse complementary chimeras, i.e. sequences erroneously assembled to contain one segment in the reverse complementary orientation as compared with the remainder of the sequence (see representative example in Supporting Information, Fig. S1a). This reverse-complemented segment led to the detection of one or more HMMs in the opposite orientation compared with the rest of the sequence. In

PLEK2 12 cases (30%), the HMMs detected a segment at either the 5′ or the 3′ end of the reverse complementary sequence that did not match any entry in GenBank; such sequences are very likely to represent chimeric unions or other sequence artefacts (see representative example in Fig. S1b). The remaining 13 cases (32.5%) contained no obvious anomaly and might represent occasional false-positive detections by individual HMMs. Importantly, though, the average HMM detection ratio between the original and the reverse complementary sequence in these 13 cases was 16 : 1, which leaves no doubt about the true orientation of the query. Considering that any 500-bp segment of a 16S sequence should have approximately 4–6 HMM detections (Hartmann et al., 2010), some sequences had lower HMM detection counts than would have been expected based on the sequence length.