Oey et al. BMC Genomics 2011, 12:315 http://www.biomedcentral.com/1471-2164/12/315 piRNAs in the database was 7,700 (defined as reads 24- 30 nt in length, the absence of 5’U and the presence of A in position 10), while that of pr19RNAs was 8,839 (defined as 19mers with an A 10nt downstream and absence of U immediately downstream of the 3’ termini). Small RNAs mapped to LINE elements To ensure that the findings were not specific to IAP, we subjected mouse LINE repeats to similar analyses. We found that the most abundant type of small RNA that mapped to the LINE reference sequence were 19mers with ~17,000 reads mapping almost exclusively to the sense strand of the repeat (Figure 2A and 2B). The com- bined number of piRNAs was ~7,500, shared equally between the strands. However, we found that the majority of piRNAs that mapped to the sense strand of the repeat had an A in position 10, indicative of s-piRNA biogenesis, while reads mapped to the antisense strand were almost exclusively p-piRNAs, recognised by U in position 1 (Fig- ure 2C). These observations support a mechanism whereby pre-existing p-piRNAs, that are antisense to endogenous LINE transcripts, target these and cause the formation of both s-piRNAs and pr19RNAs. It suggests most LINEs are transcribed in sense and thus only capable of giving rise to sense pr19RNA and sense s-piRNA, unlike IAPs, which have been shown to be transcribed from both strands [11,26]. Like the IAP-derived 19mers, the peak at 19 is clearly defined. Most 19mers in mouse spermatogenic tubules are pr19RNAs In total there were 318,960 19mers in the entire small RNA dataset that mapped to regions that were not highly repetitive (defined as having less than 6 mapped loci in the genome). They comprised 142,825 reads mapping to unique loci and 176,135 reads mapping to multiple loci. We investigated the likely origin of all these 19mers by first assigning multi-mapping reads randomly to single loci and then recording the genomic context of all the reads. Using this approach we find that in addition to being derived from retrotransposons, many 19mers are derived from piRNA clusters and refSeq genes and that the majority of these non-repeat associated 19mers also have A 10nt downstream of their 3’ termini (Figure 3). A small proportion of 19mers also overlapped with anno- tated microRNAs and with elements such as rRNAs and tRNAs. However, none of these had any obvious down- stream sequence bias and are therefore likely produced via other mechanisms such as RNA degradation (Figure 3). Notably, the small number of tRNA and rRNA-derived reads, typically considered RNA degradation products, Page 5 of 10 Figure 2 Distribution of small RNAs mapped to a LINE consensus sequence (A) The lengths of reads mapped to a LINE consensus sequence are presented. Reads mapped to the sense strand are presented above the x-axis, and reads mapped in antisense below (B) The coverage of 19mers across a 6.4 kb LINE consensus sequence is presented. Reads mapped to the sense strand are plotted above the x-axis, and reads mapped in antisense below. (C) The sequence composition of reads mapped to a LINE consensus sequence are presented. For each type of RNA the reads are separated according to their sense relative to the consensus sequence and plotted separately, as indicated. The sequence logos were extended beyond the 3’ ends of the reads (unboxed regions) to reveal any downstream sequence bias.