Authenticity of mixed seafood samples using nanopore sequencing

Maria del Socorro Toxqui Rodriguez

Seafood is the world's most highly traded food commodity. Due to the non-stop demand of the ever-increasing human population, the supply of certain species is not guaranteed, and mislabelling practices arise. Fillets and heavily processed products that cannot be easily authenticated are particularly vulnerable to fraudulent practices. In order to face this challenge, as a cost-effective, rapid, and easy handling tool, the MinION sequencer from Oxford Nanopore Technologies (ONT) represents a suitable option to assess seafood fraud. In this study, we evaluated the feasibility of MinION sequencing to authenticate mixed seafood products by (1) evaluating MinION generated barcodes from single species that have already been Sanger sequenced and (2) performing MinION sequencing of mixed samples with known compositions from different commercial species to evaluate whether all species can be detected, even when they are low abundant. Using the mitochondrial DNA gene markers COI and Cytb, 47% and 60% of the single species were unambiguously identified, 23% and 20% of the species were correctly identified but showed results for other species, and 30% and 20% of the species were wrongly identified. Concerning mixed samples, only a fraction of the species was detected regardless of the composition of the mixtures. In addition, a greater number of species was identified with the COI gene marker; however, the presence of wrongly identified species was higher than for Cytb. These results highlight that a finetuning concerning the gene marker choice, bioinformatics workflow, and database selection will be crucial for using the MinION device to investigate fraud in mixed seafood samples.

Keywords: Nanopore sequencing, MinION, Seafood fraud, seafood authentication, DNA metabarcoding.