Protocol for assembling, prioritizing, and characterizing piRNA clusters using the piRNA Cluster Builder

PIWI-interacting RNAs (piRNAs) play a critical role in safeguarding genome integrity in germ cells, ensuring fertility. Here, we provide a protocol for processing piRNA sequencing data, identifying piRNA-rich genomic regions as piRNA clusters, and preparing these clusters for downstream analyses. We describe steps for assembling piRNA cluster regions using an R-based tool, the piRNA Cluster Builder (PICB), which integrates unique and multimapping piRNA reads stepwise. The protocol allows for parameter optimizations and generates normalized outputs for prioritization of piRNA clusters. For complete details on the use and execution of this protocol, please refer to Konstantinidou et al.¹.