Thiebaut et al. BMC Genomics 2012, 13:290 http://www.biomedcentral.com/1471-2164/13/290 Page 3 of 14 Figure 1 Small RNA reads sizes distribution. Panel depicts the length distribution of redundant small RNA dataset. In the boxes are the raw numbers of small RNA reads from each size. 21 and 24-mers were the most abundance sequences. sequences mapping to the sorghum genome, and these miRNAs were classified as known miRNAs (244 unique sequences already in the miRBase) and novel miRNAs candidates (623 unique sequences) based on BLAST search against the miRBase and PMRD (Plant micro- RNA database) databases (Table 1). Overall information of all and filtered reads and numbers of miRNAs for each small RNA library are summarized in Additional file 1: Table S1. Most mature miRNAs are evolutionarily con- served between plant lineages [16]. This information enabled us to computationally predict new miRNA homo- logs or orthologs in different plant species. After the miRBase-based classification, we selected the sugarcane miRNAs conserved in sorghum. The information of pre- cursors that matched the criteria described in material and methods is showed in Additional file 1: Table S2. The length of miRNAs precursors ranged from 75 to 323 nt and their precursors fold-back structures have MFE (Minimum Free Energy) -20 to −174 kcal/mol. The MFEI (Minimum Fold Energy Index) is another parameter that was used to evaluate the novel miRNAs precursors. As reported previously by Zhang et al. (2006) [32], known plant miRNA precursors have MFEI value higher than others RNA (tRNA = 0.64, rRNA = 0.59 and mRNA = 0.65). In our analysis, more than 86% of new miRNAs had MFEI value higher than 0.7, suggesting that they are most to be likely miRNA precursors. Based on the hairpin structure profile, our sequence analysis revealed 886 precursors of novel miRNAs candi- dates (Table 1). After selection of these novel miRNA can- didates, all 623 mature miRNAs were provisory nominated like sof-miR-Seq01. Due to the high frequency of mis-annotated miRNA, the plant small RNA research community has established a set of criteria for correct annotation of miRNA [33,34]. According to these criteria, the Class I miRNA precursors have mature miRNA and miRNA* sequence found in sRNA libraries. We identified 44 bona fide precursors with miRNA/miRNA* comple- mentarity, of which 37 were unique mature miRNAs sequences. These set of microRNAs were considered high confidence and these sequences were deposited in the miRBase database. The correspondent name of these sequences in the miRBase is available in the Additional file 1: Table S3. Figure 2 shows the example of 3 precursors and their MFE values. Hairpin structures that have the lowest MFE showed the MFEI higher than 0.7; and this is strong evidence that the candidate sequence is a miRNA precursor. The structures of bona fide precursors are avail- able in Additional file 1: Figure S4. However, we cannot dismiss the other miRNAs candidates that do not fit these criteria. As demonstrated by Zhang et al. (2011) [35] there are some miRNA* that are low expressed and do not ne- cessarily appear in sequenced libraries, but the comple- mentary mature miRNA sequences were in fact miRNA. Next, we analyzed the distribution of reads by the first nucleotide at 5’end (Figure 3). Our results showed that the majority of new miRNAs candidates with 21 nt have uracile (U) at the 5’ end and the new miRNAs with 24 nt have adenine (A) in the same position. Recent studies Figure 2 Example of secondary structure of selected novel MIR genes, considered bona fide precursors. The sugarcane mature miRNA (red), miRNA* (pink) were illustrated and MFE value of stem- loop structure were also shown.