on their sequence similarity at the 59 end (nucleotides 1–10) and/ or over full length. 59 end sequences (10 nt) were considered homologous when they exhibited identity over 7 continuous nucleotides. Only interruptions implying G..U pairing were allowed within the 7 nt identity block. $70% overall similarity was the threshold used for grouping full miRNA sequences into families. miRNAs were thus classified as members of a specific family group if they met the criteria of 59 7 nt identity or $70% overall similarity with a at least one other miRNA member of the group. The sub-groups noted in supporting information contain miRNAs with more closely similar sequences ($80% overall identity or highly similar 59 ends). Expanded groupings of miRNAs exhibiting 60–69.9% sequence similarity are also included in supporting information to provide access to potentially related sequences that might be relevant to a given study. 39 similarity searches were performed with 39 end sequences (nucleotides 11-39 end) of C. elegans miRNAs. miRNA sequence analysis Analysis of mature miRNA sequences was performed using Clustal X 1.83 [87] and AlignX (a component of Vector NTi Advance 10.3.0, Invitrogen), which are both based on the Clustal W algorithm [70]. Intraspecies sequence-related miRNAs in C. elegans and D. melanogaster were evaluated by manual examination of multiple sequence alignments and 1000 bootstrapped NJ-trees. Interspecies sequence-related miRNAs were identified by manual inspection of profile alignments, in which all D. melanogaster or H. sapiens miRNA sequences were aligned against each of the 139 C. elegans miRNAs (used as reference sequence) in C. elegans-D. melanogaster and C. elegans-H. sapiens analyses, and all H. sapiens miRNA sequences were aligned against each of the 152 D. melanogaster miRNAs (reference) in the D. melanogaster-H. sapiens analysis. mir gene clusters Coordinates of mir genes in the C. elegans and D. melanogaster genomes were obtained from miRBase release 10.1, December 2007 [43–46]. mir genes were considered to form part of a cluster if they were positioned on the same DNA strand within a 2 Kb region. Diagrams were designed using Vector NTi Advance 10.3.0 (Invitrogen). Supporting Information Figure S1 Alignments of miRNA sequences conserved across species. See Table 6 and Figure 2. Grey shading identifies potential G..U pairing. Found at: doi:10.1371/journal.pone.0002818.s001 (0.06 MB PDF) Figure S2 Frequency and distribution of 59 homologous nucleotides and their correlation with overall sequence conserva- tion. A: Analysis of all 59 sequence-related miRNAs in C. elegans indicates that homologous nucleotides are mainly positioned from nucleotides 2 to 8 (Dataset S1). B: Sequence-related miRNAs with 7 or 8 homologous nucleotides at the 59 end tend to have poorer sequence similarity at the 39 end and thus weaker overall similarity than related miRNAs with 9 or 10 59 nucleotide homologies. miRNAs with 10 59 homologous nucleotides tend to have significant nucleotide similarities at the 39 end with an overall sequence identity of 70–100% (Table 1, Dataset S2) or less frequently of 60–69.9% (Dataset S3). Found at: doi:10.1371/journal.pone.0002818.s002 (0.32 MB PDF) MicroRNAs of Worms and Flies Figure S3 Sequence alignments of C. elegans miRNAs with extensive similarity at the 39 end but poor homology at the 59 end. C. elegans mature miRNAs vary in length from 18 nt to 26 nt, and thus the 39 end sequences used differed in length to some extent. Since the majority of C. elegans miRNAs are 21–23 nt long and on average 22 nt, most of the 39 end sequences varied 1–2 nt in size. Grey shading denotes potential G..U pairing. Found at: doi:10.1371/journal.pone.0002818.s003 (0.02 MB PDF) Figure S4 Average distribution of mir genes in the C. elegans and D. melanogaster genomes. Despite additional mir genes might still be discovered further concentrating genetic maps, it is worthy of note that a higher proportion of mir genes in miRBase 10.1 are located in C. elegans chromosome X (A) and in Drosophila chromosome pair 2L, 2R (B) than expected by random distribution. The expected number of mir genes was determined by dividing total number of mirs in the genome by chromosome length. Found at: doi:10.1371/journal.pone.0002818.s004 (0.39 MB PDF) Dataset S1 Homology table and sequence alignments of C. elegans miRNAs with significant identity at the 59 10 nt. Found at: doi:10.1371/journal.pone.0002818.s005 (0.39 MB DOC) Dataset S2 Homology table and sequence alignments of C. elegans miRNAs with $70% overall sequence identity. Found at: doi:10.1371/journal.pone.0002818.s006 (0.25 MB DOC) Dataset S3 Table and alignments of related C. elegans miRNAs with 60–69.9% overall sequence similarity. Found at: doi:10.1371/journal.pone.0002818.s007 (0.15 MB DOC) Dataset S4 Homology table and sequence alignments of D. melanogaster miRNAs with similar 59 ends. Found at: doi:10.1371/journal.pone.0002818.s008 (0.12 MB DOC) Dataset S5 Homology table and alignments of D. melanogaster miRNAs showing $70% overall sequence identity. Found at: doi:10.1371/journal.pone.0002818.s009 (0.08 MB DOC) Dataset S6 Table and alignments of D. melanogaster miRNA sequences with 60–69.9% similarity. Found at: doi:10.1371/journal.pone.0002818.s010 (0.15 MB DOC) Dataset S7 Homology table and alignments of C. elegans miRNAs related at the 59 end to Drosophila miRNAs. Found at: doi:10.1371/journal.pone.0002818.s011 (0.32 MB DOC) Dataset S8 Identity table and alignments of C. elegans miRNAs with $70% full sequence homology to Drosophila miRNAs. Found at: doi:10.1371/journal.pone.0002818.s012 (0.09 MB DOC) Dataset S9 Table and alignments of C. elegans and Drosophila miRNAs with 60–69.9% similarity over whole sequence. Found at: doi:10.1371/journal.pone.0002818.s013 (0.21 MB DOC) Dataset S10 Tables and alignments of C. elegans and H. sapiens miRNAs with homologous 59 ends. Found at: doi:10.1371/journal.pone.0002818.s014 (0.47 MB DOC) PLoS ONE | www.plosone.org 21 July 2008 | Volume 3 | Issue 7 | e2818