Next-Generation Sequencing as a Tool for Detailed Molecular Characterisation of Genomic Insertions and Flanking Regions in Genetically Modified Plants: a Pilot Study Using a Rice Event Unauthorised in the EU

Abstract

Precise molecular characterisation of genetic modifications integrated into the genomes of genetically modified organisms (GMOs) and of their flanking genomic regions forms a key component for the development of event-specific detection methods. In the EU, this information is of particular importance for risk management in cases where genetic modifications of unauthorised GM food, feed or seeds are detected. PCR-based chromosome walking approaches are commonly used for DNA sequence determination of the genetic modifications and of the flanking genomic regions in yet undescribed GM plants. If the plant contains complex and re-arranged modifications, sequencing and molecular characterisation are often difficult and laborious. Next-generation sequencing (NGS) of DNA is a powerful alternative tool to rapidly generate primary sequence data on the genome of so far uncharacterised sample material if pure GMO material is available. Recently, robust NGS platforms and affordable sequencing services are accessible for food and feed control laboratories. We here present a NGS-based study for whole-genome sequencing of the GM rice event LLRice62 as a proof-of-principle experiment to develop bioinformatics easy-to-use data analysis tools for rapid molecular characterisation. A total of 171,657,155 read mate pairs of approximately 75 bp each were obtained. Sequence reads belonging to the genetic modifications and their flanking genomic regions in LLRice62 were identified by bioinformatic comparison to the corresponding Oryza sativa ssp. japonica reference genome sequence using the Illumina InDel caller software and subsequent iterative mapping of retrieved NGS reads. An entire genetic modification of 1,493 bp in the genome of the LLRice62 sample material was determined and correctly mapped on chromosome 6. The determined nucleotide sequence coincides to the genetic modification described by the developer of this rice event. This study demonstrates for the first time the applicability of NGS for molecular characterisation of uncharacterised GMOs.