BFF-piRNA-seq reveals conserved processing hotspots in flies and mice

PIWI-interacting RNAs (piRNAs) protect germline genome integrity and fertility, but quantitative profiling of mature PIWI-bound RNAs remains technically challenging. Here we develop BFF-piRNA-seq, which ligates sequencing adapters directly to RNAs on immobilized PIWI complexes, eliminating preparative polyacrylamide gel electrophoresis and associated size-selection bias. Split-sample comparisons of on- and off-PIWI ligation show highly concordant length distributions and no detectable terminal sequence bias, and the method reproducibly profiles distinct PIWI-piRNA complexes in mouse and Drosophila . Eliminating gel selection recovers a broader population of Drosophila Piwi-piRNAs than conventional workflows. Across flies and mice, quantitative profiles reveal a conserved landscape across piRNA clusters in which narrow hotspots generate abundant families of sequence-related piRNAs with 5' microheterogeneity. Together, these data establish BFF-piRNA-seq as an accessible, quantitative approach for profiling bona fide piRNAs and uncover conserved principles governing piRNA selection during biogenesis and maturation.