Oey et al. BMC Genomics 2011, 12:315 http://www.biomedcentral.com/1471-2164/12/315 Page 7 of 10 Figure 5 Genes overlapped by piRNAs and 19mers (A) A piRNA cluster, transcribed from left to right and overlapped by a gene transcribed in the opposite direction, is presented (NCBI37/mm9 locus: chr10:62,120,321-62,152,434). The coverage of piRNA-sized reads (24-30nt) and 19mers (with A 10 nt downstream) is also presented in the form of wiggle plots with reads mapped from left to right above the x- axis and reads mapped from right to left below. Only uniquely mapping reads were used. The position of a gene (Stox1) is indicated with exons represented by filled boxes, arrows indicating the direction of transcription and the thin boxes representing the untranslated regions (UTRs). (B) A gene giving rise to piRNAs and and opposite sense 19mers is presented (NCBI37/mm9 locus: chr11:5,428,021-5,442,615). The coverage of piRNAs- sized reads (24-30nt) and 19mers (with A 10 nt downstream) is plotted in the form of wiggle plots with reads mapped from left to right above the x-axis and reads mapped from right to left below. Only uniquely mapping reads were used. The position of the gene (Ccdc117) is indicated with exons represented by filled boxes, arrows indicating the direction of transcription and thin boxes representing the UTRs. the abundance of s-piRNAs we find that pr19RNAs are generally more than twice as abundant as s-piRNAs (Fig- ure 6). Performing a two-sided paired Wilcoxon rank test on these values produces a p-value of 2.2 × 10-16. Of the small number of outliers that favour the s-piRNAs, most were caused by abundant reads from point sources within p-piRNA clusters and likely represent atypical p-piRNAs (lacking U in position 1) rather than s-piRNAs. The greater abundance of pr19RNAs across these non-repeti- tive loci is consistent with the observations at IAP and LINE repeats, where pr19RNAs were also found to be more abundant than s-piRNAs. Little evidence for binding of pr19RNA to Miwi or Mili We also investigated the presence of 19mers in the Miwi and Mili co-immunoprecipitated (IP) small RNA datasets from Robine et al. [7], focussing on reads mapped to IAP and LINE elements. As expected, we find that both IP libraries are enriched for ~26mers (Mili IP) and ~30mers (Miwi IP) (Figure 7). However, the peaks at 19nt from the IP libraries are lower than that from the total lysate library, suggesting these are not appreciably enriched. The peaks at 19nt observed in the IP libraries may be caused by non-specific contami- nation, rather than association with Mili or Miwi. To investigate this further we calculated the proportion of reads derived from miRNAs, snoRNAs, mitochondria and tRNAs in the IP libraries and the total lysate library (Additional file 2). RNAs derived from these RNA classes were used as they are not expected to bind Miwi or Mili directly. We find that the amount of RNA from miRNAs, snoRNAs, mitochondria and tRNAs in the IP libraries relative to the total lysate library (Additional Figure 6 Abundance of pr19RNA relative to s-piRNAs The number of piRNA-related 19mers (pr19RNA) and secondary piRNAs (s-piRNAs) overlapping 2,5kb non-overlapping tiles across the genome were plotted. Only tiles overlapping both pr19RNA and s-piRNAs in the same sense were used and tiles with abundant p-piRNAs on the same strand were excluded. Figure 7 Miwi and Mili IP small RNA derived from IAP and LINE Deep sequencing reads from co-immunoprecipitated (IP) small RNAs were mapped to an IAP consensus sequence and a LINE consensus sequence. Each plot was normalised to reads per million mappable reads in the library.