The Use of NexGen Sequencing and Junction Sequence Analysis Bioinformatics to Achieve Molecular Characterization of Crops Improved Through Modern Biotechnology

The assessment of genetically modified (GM) crops for regulatory approval currently requires a detailed molecular characterization of the DNA sequence and integrity of the transgene locus. In addition, molecular characterization is a critical component of event selection and advancement during product development. Typically, molecular characterization has relied on Southern blot analysis to establish locus and copy number, along with targeted sequencing of PCR products spanning any inserted DNA to complete the characterization process. Here we describe the use of NexGen sequencing and Junction Sequence Analysis bioinformatics in a new method for achieving full molecular characterization of a GM event without the need for Southern blot analysis. In this study, we examine a typical GM soybean line and demonstrate that this new method provides molecular characterization equivalent to the current Southern blot based method. We also examine an event containing in vivo DNA rearrangement of multiple T-DNA inserts in order to demonstrate that the new method is effective at identifying complex cases. NexGen sequencing and bioinformatics offers certain advantages over current approaches, most notably the simplicity, efficiency and consistency of the method, and provides a viable alternative for efficiently and robustly achieving molecular characterization of GM crops. The Plant Genome: Published online 15 Oct. 2012; doi: 10.3835/plantgenome2012.10.0026