Evaluation of DNA metabarcoding using Oxford Nanopore sequencing for authentication of mixed seafood products

Abstract

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) coupled with DNA (meta)barcoding 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 containing commercial seafood species in different amounts to evaluate whether all species can be detected, even when they are low abundant. Using the mitochondrial DNA gene markers Cytb and COI, 86% and 76% of the single species were correctly identified, respectively, of which 26% and 29% also showed non-target species. For the mixed samples, not all species were detected regardless of the composition of the mixtures or the marker gene used. Nevertheless, the MinION sequencing platform can be considered an additional tool to complement currently available approaches to authenticate mixed seafood samples providing that adequate measures are taken to reduce false positives and false negatives. • ONT-based metabarcoding detects 76% (COI) and 86% (Cytb) of tested seafood species. • The detection of non-target species impact interpretation of DNA metabarcoding data. • Multiple marker genes increase the detection of fish species with DNA metabarcoding.