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

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.

Anacapa Toolkit: an environmental DNA toolkit for processing multilocus metabarcode datasets

“…Stat et al, 2017). However, few metabarcode pipelines are explicitly designed to process multilocus high-throughput sequencing data (but see Arulandhu et al, 2017). As such, researchers must sort and process multiple eDNA metabarcodes independently, substantially increasing computation time with each additional metabarcode.…”

Holmes-ITS2: Consolidated ITS2 resources and search engines for plant DNA-based marker analyses

“…Due to the absence of ITS2 location annotations of some raw data, these sequences were moved to the candidate dataset first, and then a Hidden Markov Model was trained based on well-annotated ITS2 sequences to predict the potential ITS2 regions of these candidate sequences, before these ITS2 sequences could be included into our curated database. For all ITS2 sequences extracted based on the annotations, quality filter was performed in accordance with criteria as follow ( Figure 1A ): (1) length below 100 bp, (2) length above 900 bp, (3) belonging to reduplicate entries, (4) with more than three ambiguous base pairs, (5) belonging to environment samples or unclassified samples. The quality control steps filtered ITS2 entries with either low sequence quality or obscure taxonomy annotation.…”

“…Researches on plants would be carried out with the aid to the knowledge accumulated through the deep examination of the plants. On the other hand, as for researches on biodiversity and conservation of endangered fauna and flora, building accurate knowledge-base of plants is essential for their rational protection, which would also aid for preventing illegal trade of endangered plants (4). Therefore, accurate and rapid identification of plants would be essential for safe and rational utilization of plant resources and effective study and protection of plant biodiversity.…”

Botanical origin authentication of dietary supplements by DNA‐based approaches

“…Arulandhu et al. (2017) developed a multi‐locus DNA metabarcoding method based on Illumina MiSeq and 12 DNA barcode markers, which demonstrated universal applicability to species identification across a wide range of plant and animal taxa in samples containing degraded DNA. In this work, 29 animal and 17 plant reference species, representing a phylogenetically wide range of taxa (Mammalia, Actinopterygii, Malacostraca, Bivalvia, Aves, Reptilia, Amphibia, Insecta, Angiospermae, and Cycadopsida) were used to prepare complex mixtures containing two to 11 taxonomically well‐characterized species present in relative concentrations (1% to 47%).…”

“…Nevertheless, Arulandhu et al. (2017) concluded that the proposed multi‐locus DNA metabarcoding method could provide reliable and detailed data on the composition of highly complex food products and supplements.…”