A feasible method for detecting unknown GMOs via a combined strategy of PCR-based suppression subtractive hybridization and next-generation sequencing

Abstract

The rapid development of genetically modified (GM) technologies, increasing GM plant varieties and differentiated policies towards GM organisms (GMOs), make it more challenging for GMO testing nowadays. PCR-based approaches, as current routine methods, require the detailed information of transgenic events before assay. Recently reported probe-based capture strategies coupled with next-generation sequencing (NGS) offer a practical but costly way for detecting unauthorized or unknown GMOs (UGMOs) that still depend on the known GM elements as targets or start-points. Herein, we present a novel method, ‘SSH-seq’, for GMO testing based on the combination of suppressive subtractive hybridization (SSH) and NGS strategies, which has potentials for detecting UGMOs without the prior knowledge of GM cassettes. As a result, single SSH-seq experiment yielded approximately 33.8 times of reads derived from the MON87769 transgene compared to endogenous gene Lectin. For multiple SSH-seq started with eight GM soybean reference materials, part of targets were successfully enriched. Although our method might be influenced by several factors, such as the choice of degrading enzymes, GMO insertion size, sequence specificity of targets, dosage of driver DNA, NGS production, and experimental parameters, we have reasons to believe that SSH-seq will act as a powerful tool in the field of GMO testing after further optimizations, especially for UGMOs containing novel elements.