Rapid on-site universal vertebrate species identification via multi-barcode nanopore sequencing.

The growing illegal wildlife trade (IWT) threatens biodiversity and is a conduit for zoonotic disease, yet its risk of detection is low. Once processed, trafficked species are difficult to identify morphologically, and currently require DNA-based approaches that are time-consuming, costly, and lab-based. There is thus a need for a rapid, cheap, on-site method for species identification. We describe VeRIF-ID (Vertebrate Rapid In-Field Identification via DNA), a method that employs simultaneous on-site nanopore sequencing of four different mitochondrial DNA barcodes. Primers were designed to produce short amplicons to aid analysis of damaged DNA, and to be effective over a broad taxonomic range of vertebrates from lamprey to chimpanzee. Validation demonstrated species-level identification in 91% of 83 tested species, and genus/tribe-level identification of the remaining species (which are also problematic with existing approaches). DNA extraction, PCR and library preparation steps were simplified and optimised so that sampling to species identification takes <3 h for a single sample. Species components are identifiable non-quantitatively in prepared mixtures of muscle tissue from up to five species, and laboratory tests of Traditional East Asian Medicine samples reveal DNA from species including critically endangered saiga antelope and black rhinoceros. In conjunction with a portable Bento Lab device the necessary equipment and reagents are easily portable, and we apply the method to analyse seized bushmeat and fish samples within an airport customs zone, identifying mammal and fish species in 15 samples within 6 h. The initial equipment costs for VeRIF-ID are ~ $8K, and the cost per sample of ~$10-48 (depending on set-up), considerably cheaper than current conventional lab-based approaches. The method requires only basic hands-on skills. Ongoing trials with potential end-users will focus on establishing forensic reporting criteria prior to casework implementation. Future development of user-friendly bioinformatic interfaces will aim to fully democratise species identification.