Oey et al. BMC Genomics 2011, 12:315 http://www.biomedcentral.com/1471-2164/12/315 Page 6 of 10 Figure 3 Genomic annotation of all spermatogenic tubule 19mers The origin of all 19mers from the spermatogenic tubule deep sequencing dataset were deduced from overlap with elements annotated in the UCSC Genome Browser [15]. The “Clusters” category refers to 94 pachytene piRNA clusters annotated by Lau et al. [20] and includes all 19mers mapped to these that were not otherwise annotated. The category “LTR” includes all Long Terminal Repeat sequences, including the Intracisternal A-Particle. Reads that mapped to multiple loci were randomly assigned to single loci. suggest degradation is not a major contributor to the 19mer population in the dataset. Origin of non-repeat associated 19mers As expected from the analyses by Berninger et al. [12], the majority of the non-repeat associated 19mers were found to map to p-piRNA clusters. The reads mapped to both strands of these clusters, but with strand-specific differences in their downstream nucleotide composition (Additional file 1 Figure S2). Many of those in the same orientation have a 5’ U and minimal downstream sequence bias, suggesting they represent p-piRNA degradation products, while most reads in the opposite sense were found to have a bias towards A 10nt down- stream of their 3’ termini. By analysing specific piRNA clusters we find evidence of a reciprocal distribution of pr19RNAs and p-piRNAs, suggesting the antisense 19mers originate from unannotated antisense transcripts (Figure 4A). We validated the presence of one such unannotated transcript using qRT-PCR and find that the abundance of the antisense transcript is ~25% that of the sense transcript (Figure 4B). We also used the presence of pr19RNAs to locate genes that appear to be targeted by p-piRNAs. One such gene was found to overlap a p-piRNA cluster in the opposite sense with 19mers confined to an exonic region. Such a distribution suggests the 19mers originate from p-piRNA-directed cleavage of the mRNA (Figure 5A). In many other cases mRNAs were themselves found to give rise to p-piRNAs while the 19mers were found in the opposite orientation suggesting the 19mers may originate from antisense transcripts with cleavage directed by the genic p-piRNAs (Figure 5B and Addi- tional file 1 Figure S3). At all the genomic loci that were investigated pr19RNAs were found to be more abundant than s-piRNAs. To test this observation on a larger scale we investigated all regions in the mouse genome enriched for both 19mers and s-piRNAs. These regions were obtained by splitting the genome into 2.5 kb tiles and calculating ratios of dif- ferent types of uniquely mapping RNAs overlapping each tile. When plotting the abundance of pr19RNAs against Figure 4 A piRNA cluster giving rise to both piRNAs and pr19RNAs A) A bidirectional piRNA cluster is presented (NCBI37/mm9 coordinates: chr6:127,734,347-127,787,210). The coverage of piRNAs-sized reads (24-30nt) and 19mers (with A 10 nt downstream) is visualised by 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 are plotted. The position of repetitive elements, adapted from the UCSC Genome Browser [15], is indicated below the plots. The approximate position of two PCR targets used for strand-specific real-time quantitative reverse transcription PCR (qRT-PCR) is also indicated. B) The abundance of the piRNA precursor transcript relative to that of an overlapping antisense precursor transcript is presented. The fold- difference was estimated by strand-specific qRT-PCR targeting two separate loci, as indicated.