Authenticity of mixed seafood samples using nanopore sequencing

Seafood plays an important role in the human diet. Fish stocks are under pressure due to the non-stop demand of the ever-increasing human population. As a consequence, the supply of certain fish species is not guaranteed and the substitution of some fish species for other closely related species with a lower value in the global marketplace is frequent. The intentional adulteration of seafood for financial advantage, also known as seafood fraud, has become a worldwide problem. In addition to consumer fraud, food mislabelling has consequences in human health as toxic species may enter the market and allergy issues may arise. Fillets and heavily processed seafood products that lack reliable morphological features cannot be easily authenticated by the eye and are thus particularly vulnerable to fraudulent practices.
The genetic assessment of seafood ingredients generally relies on standard DNA barcodes for animals. However, this is challenging when it comes to mixed samples, and here, high-throughput amplicon sequencing represents a powerful tool to authenticate complex food products. As a cost-effective, rapid, and easy handling tool for characterizing mixed samples, the MinION sequencer from Oxford Nanopore Technologies (ONT) represents a suitable option to assess seafood fraud even directly in the field.
In this study, we assess the feasibility of MinION sequencing to authenticate mixed seafood products. DNA was extracted from 16 commercial fish species which were then pooled to obtain five mixtures containing only closely related species (cod, salmon, herring, sardine and flatfish), a sixth mix was made containing only distantly related species and the last mix contained all 16 species. The seven mixes served as template for PCR amplification of the COI and Cytb gene markers, which are commonly used for fish DNA barcoding. The PCR products were processed according to the Ligation Sequencing Kit (SQK-LSK109) in conjunction with the Native Barcoding Expansion (EXP-NBD104) kit and sequenced using an R9.4.1 flow cell. Sequences were quality filtered and bioinformatically processed using Decona, a bioinformatic pipeline that creates consensus sequences from mixed (metabarcoding) samples.
In all mixes and for both COI and Cytb, consensus sequences were retrieved for 12 out of the 16 fish species. Only the flatfish (Solea solea, Limanda limanda, Platichthys flesus, and Pleuronectes platessa) were not detected. This may be linked to our bioinformatics workflow, sample processing or to the sequencing errors that are generated when working with mixed samples. In order to face this challenge, an additional case study involved sequencing of 21 single species that are commonly mislabelled, including the flatfish species. In addition, homogenized tissue mixtures and mixtures of fish DNA extracts at different concentrations were included. Results of these three additional tests will allow to evaluate and finetune the reliability of MinION sequencing to investigate fraud in mixed seafood samples.