Species identification in mixed tuna samples with next-generation sequencing targeting two short cytochrome b gene fragments

- cytb targeted NGS allowed the identification of fish species in mixed tuna samples.
- Sequence recovery from prepared tuna mixtures was very precise.
- Skipjack tuna was overrepresented compared to Thunnus species.
- Phylogenetic grouping of recovered sequences was suitable for tuna tin analysis.
- This technology is a promising tool for tuna tin authentication in the future.

Conventional Sanger sequencing of PCR products is the gold standard for species authentication of seafood products. However, this method is inappropriate for the analysis of products that might contain mixtures of species, such as tinned tuna. The purpose of this study was to test whether next-generation sequencing (NGS) can be a solution for the authentication of mixed products. Nine tuna samples containing mixtures of up to four species were prepared and subjected to an NGS approach targeting two short cytochrome b gene (cytb) fragments on the Illumina MiSeq platform. Sequence recovery was precise and admixtures of as low as 1% could be identified, depending on the species composition of the mixtures. Duplicate samples as well as two individual NGS runs produced very similar results. A first test of three commercial tinned tuna samples indicated the presence of different species in the same tin, although this is forbidden by EU law.