Read-mapping signatures for molecular characterization of transgenic DNA insertions in paired-end Illumina data

Transgenic crops undergo rigorous safety assessments prior to commercialization, with molecular characterization serving as a critical component of regulatory review. This process establishes the identity, copy number, sequence integrity, absence of unintended foreign DNA, and insert stability across breeding generations. While whole-genome sequencing (WGS) has emerged as a powerful alternative to Southern blotting, the lack of accessible interpretation frameworks can be an entry barrier to those who wish to understand this modernized experimental setup. We developed an analytical workflow based on mapped-read signatures to characterize T-DNA (transfer DNA) insertions using short-read WGS data. Simulated Illumina paired-end datasets representing diverse transformation outcomes were generated and analyzed to define five informative read classes, which when observed mapped to a reference transformation construct provide distinct signatures indicating transformation outcomes. These signatures were applied to identify insertion boundaries, copy number, structural anomalies, and potential contamination. Mapped-read signatures can reliably distinguish single-copy inserts, multiple insertions, backbone co-integrations, and structural rearrangements, aided by coverage profiles and mate-pair orientations. We present representative examples and a practical interpretation to guide practitioners new to WGS-based molecular characterization and regulators assessing these data. This framework standardizes interpretation of short-read paired-end WGS data for molecular characterization without prescribing specific software. The platform-agnostic approach ensures broad applicability while enhancing transparency in regulatory assessments.