A duplex sequencing approach for high-sensitive detection of genome edited plants

New genomic techniques (NGT) are able to alter genomes at specified sites often generating single nucleotide variants (SNV) or short insertions/deletions. The detection of these products by droplet digital PCR (ddPCR) and real-time PCR methods at the level required by the EU GMO legislation is challenging. In fact, the development of such methods requires prior knowledge of the mutation and relies on ad hoc approaches. In this paper, we have evaluated a targeted high-throughput massive parallel sequencing approach. It uses unique molecular identifiers (UMIs) for the quantification of the variant alleles and duplex sequencing to confirm a mutation on both strands to avoid PCR artefacts or sequencing miss-calls. We tested the strategy on a set of tomato lines carrying small genomic alterations generated by Clustered Regularly Interspaced Short Palindromic Repeats-associated protein 9 (CRISPR/Cas9) and we were able to detect true mutations down to a 0.1 % level (in copy number) and discriminate them from PCR amplification and sequencing errors. We have also assessed the performance of the duplex sequencing approach for the determination of the mutant DNA content in samples containing different NGT lines. To our knowledge, this is the first attempt to use a duplex sequencing approach with no a priori knowledge of the mutation type to address the analytical challenges inherent to the quantification of short DNA alterations resulting from NGTs.