Nucleic Acids Research, 2011, Vol. 39, No. 15 6599 Figure 1. piRNA and pilRNA features in ﬂy tissues. (A) Length distribution of all mapped sequence reads in ﬂy tissues. Here (and further), the labels indicate: OSS, ovarian somatic sheet cells; EMB, embryo; OVY, ovary; HED, head; IMD, imaginal discs. For a full sample description, see Supplementary Table S13. (B) Length distribution of all mapped sequence tags (sequence reads after collapsing identical sequences). (C and D) Length distributions of sequence reads and tags, excluding reads mapped to known ncRNA, respectively. Note that 50 uridine bias is one known property of primary piRNA. (E) Proportions of complementary small RNA reads (24–32 nt) at different offsets. Excess of complementary reads at offset equal 10 nt indicates the ping-pong model signature. (F) The stand (S) and the 50 uridine (U) bias of sequence reads’ clusters based on 24–32 nt long sequences. (G) The upper panel shows genomic context of small RNA clusters (24–32 nt) identiﬁed in ﬁve ﬂy tissues using publicly available data sets (11,21,29,30–33). The lower panel shows relative abundance of uniquely mapped small RNA (24–32 nt) among four previously characterized ﬂy TE master loci (11). categories: (i) putative primary piRNA—small RNA that have U as the ﬁrst nucleotide and (ii) putative secondary piRNA—the rest of the sequences. Sequences that have both U at position 1 and A at position 10 were excluded from the putative primary piRNA group because these sequences could not be assigned to one of the groups un- ambiguously. Next, we investigated the ping-pong model signature by checking the complementarity, again at dif- ferent offsets, between putative primary piRNA and putative secondary piRNA that originated from the opposite strand (of the primary piRNA) and that were within 20 nt from the 50-end of the primary piRNA. The relative complementary rate at each offset value was calculated by normalizing by the sum of complementary rate at each offset. We then determined the signal to noise ratio (SNR) for each offset position, calculated as a ratio of complementary rate at a given offset to the mean value of complementary rates at the other 19 offset positions. If the SNR at position 10 had the highest SNR value and was >4, and the ratio between SNR at the 10th position and the second highest SNR was >3, we classiﬁed this tissue as showing the ping-pong ampliﬁcation model signature. We further investigated the ping-pong model signature based on small RNA sequences (24–32 nt, excluding ncRNA) originating within identiﬁed clusters, using the same procedure (Supplementary Tables S1, S4, S8). If the ping-pong model signature based either on small RNA sequences (24–32 nt, excluding ncRNA) or small RNA sequences (24–32 nt, excluding ncRNA) originated from identiﬁed small RNA clusters ﬁt the