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. 2017 Oct 1;6(10):1-18.
doi: 10.1093/gigascience/gix080.

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

Alfred J Arulandhu  1   2 , Martijn Staats  1 , Rico Hagelaar  1 , Marleen M Voorhuijzen  1 , Theo W Prins  1 , Ingrid Scholtens  1 , Adalberto Costessi  3 , Danny Duijsings  3 , François Rechenmann  4 , Frédéric B Gaspar  5 , Maria Teresa Barreto Crespo  5 , Arne Holst-Jensen  6 , Matthew Birck  7 , Malcolm Burns  8 , Edward Haynes  9 , Rupert Hochegger  10 , Alexander Klingl  11 , Lisa Lundberg  12 , Chiara Natale  13 , Hauke Niekamp  14 , Elena Perri  15 , Alessandra Barbante  15 , Jean-Philippe Rosec  16 , Ralf Seyfarth  17 , Tereza Sovová  18 , Christoff Van Moorleghem  19 , Saskia van Ruth  1   2 , Tamara Peelen  20 , Esther Kok  1
Affiliations

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

Alfred J Arulandhu et al. Gigascience. .

Abstract

DNA metabarcoding provides great potential for species identification in complex samples such as food supplements and traditional medicines. Such a method would aid Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) 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 forensic wildlife species identification and to evaluate the applicability and reproducibility of this approach across different laboratories. A DNA metabarcoding method was developed that 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 species in samples containing degraded DNA. The DNA metabarcoding method was developed based on Illumina MiSeq amplicon sequencing of well-defined experimental mixtures, 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. 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-listed species. The method can provide improved resolution for species identification, while verifying species with multiple DNA barcodes contributes to an enhanced quality assurance.

Keywords: CITES; COI; Customs agencies; DNA metabarcoding; Endangered species; Traditional medicines; cyt b; matK; mini-barcodes; rbcL.

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Figures

Figure 1:
Figure 1:
Schematic representation of the CITESspeciesDetect pipeline.
Figure 2:
Figure 2:
The percentage of QC reads assigned to DNA barcodes for samples EM10, EM10R, EM11, and EM11R of the pilot study.
See this image and copyright information in PMC

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