Gunaratne et al. BMC Genomics 2011, 12:277 http://www.biomedcentral.com/1471-2164/12/277 Page 3 of 14 Figure 1 Pipeline with yields for analysis of putative novel miRNAs. 52 small RNA sequences did not align to miRBase reference sequences and were assessed for miRNA potential. 48 sequences passed the minimum criteria and were categorized into three groups according to strength of evidence (sequences are color-coded in Additional File 1, Table S3, as indicated). Seven (7) are confirmed novel miRNAs since they had all the characteristics of known miRNAs and in addition also had a less abundant miR* sequence that maps on the opposite side of the stem from the putative novel miRNA. These are labelled green in Additional File 1, Table S3. Twenty-one (21) putative novel miRNAs are highly confident (labelled blue) since they also shared characteristics of known miRNAs but no sequence was found aligning to the miR* region. Given that the miR and miR* sequences for most known miRNAs have vastly different copy numbers such that the miR* sequence is sometimes not found, the highly confident candidates are also highly likely to be genuine novel miRNAs, Twenty (20) candidates (labelled grey) had a subset of the characteristics of known miRNAs but not all and therefore were deemed potential candidates that require more evidence.