BaseClear and RIKILT (Wageningen UR) have recently collaborated to develop innovative methods for GMO traceability. The work was coordinated by Jeroen van Dijk and Marleen Voorhuijzen (RIKILT) within the context of the FP7 European Decathlon project. The preliminary results were presented by Adalberto Costessi (Product Manager NGS, BaseClear) at the Oxford Nanopore Community Meeting in New York in December 2016, and by Marleen Voorhuijzen at the Oxford Nanopore London Calling event in May 2017. A scientific article has been recently submitted for publication.
The EU has strict regulation on the import and production of genetically modified (GM) food and feed and it requires a stringent safety approval before a GM plant can be introduced in the environment or sold on the market.
For all GM plants that have been authorized, specific validated qPCR identification and detection assays are available. These are based on the knowledge of the inserted DNA sequences and the locus where these sequences were introduced in the plant genomes. However, the growing number of biotech crops with novel genetic elements increasingly complicates GMO detection by qPCR, since many different assays must be performed and qPCR does not support a high level of multiplexing.
Importantly, there are no specific detection assays available for GMO plants that have not been (yet) authorized in the EU (UGMO – unauthorized GMO), and this poses significant limitations to the present analysis of GMO traceability.
OBJECTIVE AND APPROACH
The objective of this project was to develop an innovative genome-walking approach combined with NGS sequencing in order to generate sequence information of the GMO elements as well as the adjacent genomic regions. This approach would allow analyses of both known as well as unknown GMOs with the same assay. The vast majority of GMOs, including UGMOs, contains one or more known ‘elements’ (i.e. 35S promoter). We used these elements as starting points to reach either (or both) insertion points in UGMOs, in complex food and feed samples. The enrichment protocol used for this project is shown in the figure below:
NGS SEQUENCING ON THE MINION
For a first proof-of-concept experiment we used a test sample containing the well-characterized MON810 GMO material. We enriched the sample with an optimized protocol, using 1 GM element P-35S. Multiple conditions were tested for MinION sample preparation and MinION sequencing, i.e. amount of input DNA, library prep kit and chemistry version. The preliminary analysis showed that the expected GM sequence could be identified successfully in the MinION datasets!
The conclusion of this project is that MinION sequencing of enriched test samples identified successfully the expected GMO event, even with very low input. The next step is to analyse unknown samples and test complex mixtures.
- Volpicella M, Leoni C, et al. Genome Walking by Next Generation Sequencing Approaches Biology 2012, 1, 495-507
- Liang C, van Dijk JP, et al. Detecting authorized and unauthorized genetically modified organisms containing vip3A by real-time PCR and next-generation sequencing. Anal Bioanal Chem. 2014 Apr;406(11):2603-11
- Spalinskas R, Van den Bulcke M et al. LT-RADE: An Efficient User-Friendly Genome Walking Method Applied to the Molecular Characterization of the Insertion Site of Genetically Modified Maize MON810 and Rice LLRICE62 Food Anal. Methods (2013) 6:705–713