T-DNA Analyzer: A Long-Read Sequencing Pipeline for Characterizing T-DNA Insertion Sites in Transgenic Crops

Molecular characterization of the transferred DNA (T-DNA) insertion sites is required for the safety assessment of genetically modified (GM) crops, yet conventional PCR-based methods are labor-intensive and limited in their ability to resolve complex structural variations. We present T-DNA Analyzer, an integrated bioinformatics pipeline that transforms long-read sequencing data (PacBio HiFi or Oxford Nanopore) into a comprehensive insertion site report. The pipeline implements a host-derived read filter that subtracts host-homologous vector regions to eliminate false-positive chimeric read calls; a multi-segment fusion detection algorithm that resolves complex T-DNA integration architectures; and a deletion gap gene impact analysis that identifies genes affected by host genome deletions at the integration site. Validation on maize and cotton datasets demonstrated that the host-derived filter excluded 86.4% of false-positive reads while retaining all true chimeric reads, and the fusion detection algorithm successfully reconstructed a two-copy tandem T-DNA repeat within a single long read. T-DNA Analyzer provides automated, reproducible molecular characterization designed to support regulatory molecular characterization and is freely available as open-source software.