Development and validation of a multi-locus DNA metabarcoding method to identify endangered species in complex...

Area of Science:

• Forensic science
• Molecular biology
• Conservation genetics

Background:

• DNA metabarcoding offers powerful species identification in complex matrices like food supplements and traditional medicines.
• Accurate species identification is crucial for enforcing regulations against illegal wildlife trade, particularly for CITES-listed species.
• Existing methods may face challenges with degraded DNA or complex mixtures, necessitating robust and reproducible techniques.

Purpose of the Study:

• To develop and validate a multi-locus DNA metabarcoding method for forensic wildlife species identification.
• To assess the method's applicability, reproducibility, and sensitivity across diverse laboratories.
• To support Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) enforcement by detecting endangered species in trade.

Main Methods:

• Development of a DNA metabarcoding approach utilizing 12 universal DNA barcode markers.
• Application of Illumina MiSeq amplicon sequencing on well-defined experimental mixtures.
• Creation of a user-friendly bioinformatics pipeline with a web interface for data analysis.
• International validation trial involving 16 laboratories to assess performance.

Main Results:

• The developed method demonstrated high reproducibility and sensitivity in an international validation trial.
• Species present at 1% dry weight in mixtures were accurately identified.
• The multi-locus approach provided reliable data on the composition of complex food products.
• The method successfully identified CITES-listed species, confirming its forensic applicability.

Conclusions:

• The advanced multi-locus DNA metabarcoding method provides reliable and detailed species identification in complex samples.
• This validated technique enhances the quality assurance of species identification through multiple DNA barcodes.
• The method is suitable for forensic applications, aiding in the combat of illegal wildlife trade and CITES enforcement.