TruePaiR: Software for the Accurate Identification of Complementary piRNA Read Pairs in High-Throughput Sequencing Data

piRNAs and their biogenesis pathways are well-conserved in Metazoans (Grimson et al. 2008). piRNAs have been implicated in transcriptional, post-transcriptional, and translational regulation (Grivna et al. 2006; Lin & Yin 2008; Brennecke et al. 2008; Brennecke et al. 2007; Aravin et al. 2007). We analyze the signatures of a critical process in the primary and secondary mechanism of piRNA biogenesis, referred to as the amplification loop. The presence of U-1 and A-10 bias within piRNA populations is an indicator, but not an absolute measure of piRNA amplification. By further considering imperfect and perfect sequence complementarity within the first ten base pairs of piRNAs, the active site promoting secondary piRNA biogenesis, we developed practical and statistically powerful metrics to observe relative piRNA amplification. TruePaiR is a fast and effective general software tool to assess the relative utilization of piRNA amplification in high throughput sRNA sequencing data. The results of TruePaiR runs in seven species and five tissues serve as a benchmark for meaningful context of piRNA amplification. The TruePaiR metrics provide foundational data regarding the in terms of species specificity, tissue specificity, as well as the relative participation based upon origin-based piRNA subsets regarding piRNA amplification. The low degree of variability of same sample TruePaiR runs allows for metric reliability, reproducibility, as well as the ability to detect subtle differences in piRNA amplification within and between species and tissues. Given that TruePaiR serves as an effective and consistent metric of piRNA amplification across species, it can represent a new, meaningful standard in the degree of piRNA amplification in a specific organism and tissue that is or is not expected to undergo piRNA amplification.