With the continual development of genetically modified (GM) crops, it has become necessary to develop detailed and effective molecular characterization methods to select candidate events from a large pool of transformation events. Relative to traditional molecular analysis methods such as the polymerase chain reaction (PCR) and Southern blot hybridization, next generation sequencing (NGS) technology for whole-genome sequencing of complex crop genomes had proven comparatively useful for in-depth molecular characterization. In this study, four transformation events, including one in Bacillus thuringiensis (Bt)-resistant rice, one in resveratrol-producing rice, and two in beta-carotene-enhanced soybeans, were selected for molecular characterization. To merge NGS analysis and Southern blot-hybridization results, we confirmed the transgene insertion sites, insertion construction, and insertion numbers of these four transformation events. In addition, the read-coverage depth assessed by NGS analysis for inserted genes might provide consistent results in terms of inserted T-DNA numbers in case of complex insertion structures and highly duplicated donor genomes; however, PCR-based methods can produce incorrect conclusions. Our combined method provides an effective and complete analytical approach for whole-genome visual inspection of transformation events that require biosafety assessment.

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• Molecular Characterization of CRISPR-Cas9-Edited Rice Across Generations and Associated Technical Challenges in Nucleotide Editing Tracing
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  Plant Breeding and Biotechnology.2025;[Epub]     CrossRef
• Combining Nanopore and Illumina Sequencing Permits Detailed Analysis of Insertion Mutations and Structural Variations Produced by PEG-Mediated Transformation in Ostreococcus tauri
  Julie Thomy, Frederic Sanchez, Marta Gut, Fernando Cruz, Tyler Alioto, Gwenael Piganeau, Nigel Grimsley, Sheree Yau
  Cells.2021; 10(3): 664.     CrossRef
• Comparative transcriptome profiling of different tissues from beta-carotene-enhanced transgenic soybean and its non-transgenic counterpart
  Yang Qin, Hee-Jong Woo, Kong-Sik Shin, Myung-Ho Lim, Seong-Kon Lee
  Plant Cell, Tissue and Organ Culture (PCTOC).2020; 140(2): 341.     CrossRef