International interlaboratory validation trial of a multi-target DNA metabarcoding approach to identify endangered species in complex and wildlife forensic samples

Background: Identification of endangered species by customs and regulatory agencies from products such as food supplements or Traditional Medicines (TMs) is challenging due to the complex nature of the samples. Here, we developed a standard operating procedure (SOP) for a multi-target metabarcoding approach including a CITESspeciesDetect pipeline and web interface. The reproducibility of the approach was validated across 16 laboratories . Results: First an optimal DNA isolation method for experimental samples and TMs, and a single PCR condition for 12 universal barcode and mini-barcode markers was identified. The developed multi-targeting metabarcoding panel was successfully evaluated in a pilot study with 15 unique experimental samples, based on the results a SOP was established. In the validation study across laboratories, comprising 8 complex mixtures and 2 TMs, the approach was found to be highly reproducible across and sensitive enough to detect low abundance species (1%) in all the experimental samples. In case of the TMs, only a few ingredients listed on the labels could be identified reproducibly and most identified taxa were unlabelled ingredients. In the analysis, revealed that the combination of multiple barcode markers is necessary for optimal resolution in species identification . The CITESspeciesDetect pipeline and web interface was proven to be user-friendly, robust and accurate. Conclusion: We developed and validated an adequate multi-target metabarcoding to identify endangered species in complex seized wildlife samples. Furthermore, the approach improved resolution and sensitivity for multi species identification, a sample-to-answer procedure is available to be applied in routine laboratory setups.