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

The assessment of genetically modifi ed (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 polymerase chain reaction products spanning any inserted DNA to complete the characterization process. Here we describe the use of next generation (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 [Glycine max (L.) Merr.] 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 transfer DNA inserts to demonstrate that the new method is effective at identifying complex cases. Next generation sequencing and bioinformatics offers certain advantages over current approaches, most notably the simplicity, effi ciency, and consistency of the method, and provides a viable alternative for effi ciently and robustly achieving molecular characterization of GM crops. MOLECULAR CHARACTERIZATION is a key step in the assessment of genetically modifi ed (GM) crops for regulatory approval. Current characterization methods are designed to accurately establish a number of important properties of the inserted DNA in the GM crop’s genome and this characterization is a key step in the production of improved GM crops. Th ese characterizations are required for selection of the events with the most favorable molecular profi le throughout research activities (Heck et al., 2005; Vaughn et al., 2005; Cerny et al., 2010) and are also performed as a last step before commercialization, where it is a crucial part of the comparative assessment process endorsed by Codex and used by most global regulatory organizations (Codex Alimentarius, 2003). Molecular characterization of inserted DNA and associated native fl anking sequences consists of determining the number of insertion sites, the insert copy number at each insertion site, the DNA sequence of each inserted DNA, and the sequence of the native locus at each site. Current methods also establish a description of any genetic rearrangements that may have occurred at the insertion site as a consequence of transformation and demonstrate the absence of unintended plasmid DNA (i.e., non-transfer DNA [T-DNA] backbone sequence) within the GM event. Generational stability analysis, which demonstrates the stable heritability of inserted DNA Published in The Plant Genome 5:149–163. doi: 10.3835/plantgenome2012.10.0026 © Crop Science Society of America 5585 Guilford Rd., Madison, WI 53711 USA An open-access publication All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher. D. Kovalic, C. Garnaat, Y. Yan, J. Groat, A. Silvanovich, L. Ralston, M. Huang, Q. Tian, A. Christian, N. Cheikh, J. Hjelle, S. Padgette, and G. Bannon, Monsanto Company, 800 N. Lindbergh Blvd., St. Louis, MO 63167; L. Guo, Bayer Crop Science, 2 T.W. Alexander Dr., RTP, NC 27709. Received 11 Oct. 2012. *Corresponding author ([email protected]). Abbreviations: ABI, Applied Biosystems, Inc.; CTAB, hexadecyltrimethylammonium bromide; EDTA, ethylenediaminetetraacetic acid; GM, genetically modifi ed; JSA, junction sequence analysis; MW, molecular weight; NexGen, next generation; PCI, phenol:chloroform:isoamyl alcohol; PCR, polymerase chain reaction; T-DNA, transfer DNA; TE, Tris-EDTA; WGS, whole-genome shotgun.