Mechanism-Guided Bioinformatic Analysis of Non-LTR Retrotransposon Insertions: From Molecular Archaeology to De Novo Experimental Systems

Non-long-terminal-repeat (non-LTR) retrotransposons insert into the genome by priming cDNA synthesis directly from the target site in a process termed target-primed reverse transcription, but the subsequent steps that complete insertion remain poorly understood. In contrast to the relatively uniform products of integrase-mediated LTR retrotransposon integration, non-LTR retrotransposon insertions are structurally heterogeneous, complicating their analysis. Yet this heterogeneity can reveal variation in the underlying insertion reaction when interpreted in an appropriate mechanistic context. Here, we present a framework for mechanism-guided bioinformatic analysis of non-LTR retrotransposon insertions, based on the premise that distinct features of insertion architecture and genomic context report on different biochemical steps of the insertion process. Drawing on historical and recent studies, we identify best practices for sequencing and bioinformatic analysis and consider how extant genomic insertions and experimentally generated de novo insertions provide complementary mechanistic information while imposing different inferential biases. We envision that standardized principles for mechanism-guided analysis will help resolve poorly understood steps of insertion completion, enable comparative investigation across retrotransposons and hosts, and improve evaluation of emerging retrotransposon-based genome-engineering systems.