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

Area of Science:

• Agricultural Biotechnology
• Genomics
• Bioinformatics

Background:

• Molecular characterization of transferred DNA (T-DNA) insertion sites is crucial for genetically modified (GM) crop safety assessments.
• Conventional PCR-based methods are inefficient and struggle with complex structural variations at T-DNA insertion sites.

Purpose of the Study:

• To develop an integrated bioinformatics pipeline, T-DNA Analyzer, for comprehensive molecular characterization of T-DNA insertion sites using long-read sequencing data.
• To improve the accuracy and efficiency of T-DNA insertion site analysis for GM crop safety evaluations.

Main Methods:

• T-DNA Analyzer utilizes PacBio HiFi or Oxford Nanopore long-read sequencing data.
• It incorporates a host-derived read filter to eliminate false-positive chimeric reads.
• A multi-segment fusion detection algorithm resolves complex T-DNA integration architectures.
• Deletion gap gene impact analysis identifies host genes affected by deletions at the integration site.

Main Results:

• The host-derived filter successfully excluded 86.4% of false-positive reads while retaining true chimeric reads.
• The fusion detection algorithm accurately reconstructed a two-copy tandem T-DNA repeat within a single long read.
• Validation was performed on maize and cotton datasets.

Conclusions:

• T-DNA Analyzer provides automated, reproducible molecular characterization of T-DNA insertion sites.
• The pipeline supports regulatory safety assessments of GM crops.
• T-DNA Analyzer is available as open-source software.