Development and international validation trial of an advanced, multi-locus DNA metabarcoding metho to identify endangered species in complex samples

Background: DNA metabarcoding, which involves Next-Generation Sequencing (NGS) of DNA barcodes, holds great promise for species identification in complex samples such as food supplements and Traditional Medicines (TMs). Such method would aid CITES (the Convention on International Trade in Endangered Species of Wild Fauna and Flora) enforcement officers to combat wildlife crime by preventing illegal trade of endangered plant and animal species. The objective of this research was to develop a multi-locus DNA metabarcoding method for wildlife forensic species identification and to evaluate the applicability and reproducibility of the this approach across different laboratories. Results: The DNA metabarcoding method developed in this study makes use of 12 DNA barcode markers that have demonstrated universal applicability across a wide range of plant and animal taxa, and that facilitate the identification of plant and animal species in highly processed samples containing degraded DNA. The DNA metabarcoding method was developed on the basis of NGS data generated for 15 well-defined experimental mixtures using Illumina MiSeq technology, for which a bioinformatics pipeline with user-friendly web interface was developed. The performance of the DNA metabarcoding method was assessed in an international validation trial by 16 laboratories, in which the method was found to be highly reproducible and sensitive enough to identify species present in a mixture at 1% dry weight content. Conclusion: The advanced, multi-locus DNA metabarcoding method assessed in this study provides reliable and detailed data on the composition of complex food products, including information on the presence of CITES species. The method provides improved resolution for species identification, while verifying species with multiple DNA barcodes contributes to enhanced quality assurance.