Oey et al. BMC Genomics 2011, 12:315 http://www.biomedcentral.com/1471-2164/12/315 Page 4 of 10 Figure 1 Features of small RNAs mapped to an IAP consensus sequence (A) The lengths of small RNAs that mapped to IAP are presented. Reads mapped to the sense strand are presented above the x-axis, and reads mapped in antisense below. (B) The sequence composition of the IAP-derived 19mers and piRNAs were obtained from sequence logos. The reads were separated by their orientation relative to an IAP reference sequence and plotted separately, as indicated. The sequence logos were also extended beyond the 3’ ends of the reads (unboxed regions) to reveal any downstream sequence bias. (C) The density of 19mer RNAs mapped to IAP in sense (only reads with an A 10nt downstream and absence of U immediately downstream of the 3’ terminus were used), secondary piRNAs mapped to IAP in sense (24-30nt RNAs with A in position 10 and absence of 5’ U) and primary-piRNAs mapped to IAP in antisense (24-30nt RNA with 5’ terminal U and absence of A in position 10), are plotted. Only the 5’ ends of the reads were used to calculate the density. (D) The abundances of IAP-derived p-piRNAs and s-piRNAs, at varying distances from IAP-derived piRNA-related 19mers (pr19RNAs) 5’ ends, were tallied and are presented as proportions of the total RNA count. 19mer mapped to IAP in sense (configuration illustrated in Figure 1D). It did not escape our notice that some 19mers had a strong bias towards U immediately downstream of their 3’ ends (Figure 1B). We found that many of these 19mers were positionally locked to the 5’ end of same- sense p-piRNAs while they overlapped with opposite sense s-piRNA (Additional file 1 Figure S1A). This sug- gests that this latter group is formed from enzymatic cleavage of IAP guided by s-piRNAs, thus causing the release of a 19mer with a nucleotide bias characterised by a U immediately downstream of the 3’ terminus, instead of an A 10nt downstream. We tallied the num- ber of 19mers that were either immediately adjacent to a piRNA-sized read, or antisense to one, in the two pos- sible configurations indicated in Figure 1D and Addi- tional file 1 Figure S1A, but disregarding whether the piRNAs where p-piRNAs or s-piRNAs. We found that out of the 9,648 19mers mapped to the sense strand, 8,861 (91%) were found with at least one piRNA in such a configuration, while out of 8,139 19mers mapped in antisense, 7,238 (89%) were arranged in this manner. The remaining IAP-derived 19mers have minimal down- stream sequence bias, and likely represent RNA degra- dation products. As the 19mers seem likely to be formed via a piRNA-dependent mechanism, we call them piRNA-related 19mers, or pr19RNAs. We also investigated the full complement of small RNAs that mapped to any mouse genomic IAP element in our database, as many were not mappable to the con- sensus IAP sequence. This increased the number of IAP-derived reads two-fold but had only a minor effect on the distribution of 19mers relative to piRNAs (Addi- tional file 1 Figure S1B and S1C). The number of s-