Targeted MinION sequencing of transgenes

Abstract

AbstractThe presence of genetically modified organisms (GMO) is commonly assessed using real-time PCR methods targeting the most common transgenic elements found in GMOs. Once the presence of GM material has been established using these screening methods, GMOs are further identified using a battery of real-time PCR methods, each being specific of one GM event and usually targeting the junction of the plant genome and of the transgenic DNA insert. If, using these specific methods, no GMO could be identified, the presence of an unauthorized GMO is suspected. In this context, the aim of this work was to develop a fast and simple method to obtain the sequence of the transgene and of its junction with plant DNA, with the presence of a screening sequence as only prior knowledge. An unauthorized GM petunia, recently found on the French market, was used as template during the development of this new molecular tool. The innovative proposed protocol is based on the circularization of fragmented DNA followed by the amplification of the transgene and of its flanking regions using long-range inverse PCR. Sequencing was performed using the Oxford Nanopore MinION technology and a bioinformatic pipeline was developed.