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.

AGENZIA DELLE DOGANE E DEI MONOPOLI Laboratori e servizi chimici Laboratorio Chimico di Genova 16126 Genova Via Rubattino n 6 Italy

Austrian Agency for Health and Food Safety Spargelfeldstrasse 191 1220 Vienna Austria

Baseclear B 5 Einsteinweg 5 2333 CC Leiden The Netherlands

Biolytix AG Benkenstrasse 254 4108 Witterswil Switzerland

CREA SCS sede di Tavazzano Laboratorio via Emilia Km 307 26838 Tavazzano Italy

Crop Research Institute Department of Molecular Genetics Drnovská 507 161 06 Prague Czech Republic

Dutch Customs Laboratory Kingsfordweg 1 1043 GN Amsterdam The Netherlands

Eurofins GeneScan GmbH Engesserstrasse 4 79108 Freiburg Germany

Fera Sand Hutton York YO41 1LZ UK

Food Quality and Design Group Wageningen University and Research P O Box 8129 6700 EV Wageningen The Netherlands

Generalzolldirektion Direktion 9 Bildungs und Wissenschaftszentrum der Bundesfinanzverwaltung Dienstort Hamburg Baumacker 3 D 22523 Hamburg Germany

GenoStar Bioinformatics Solutions 60 rue Lavoisier 38330 Montbonnot Saint Martin France

iBET Instituto de Biologia Experimental e Tecnológica Apartado 12 2780 901 Oeiras Portugal

Laboratory of Customs and Excises Blijde Inkomststraat 20 B 3000 Leuven Belgium

LGC Queens Road Teddington Middlesex TW11 0LY UK

Livsmedelsverket Att Lisa Lundberg Strandbodgatan 4 SE 75323 Uppsala Sweden

Norwegian Veterinary Institute Ullevaalsveien 68 P O Box 750 Sentrum 0106 Oslo Norway

RIKILT Wageningen University and Research P O Box 230 6700 AE Wageningen The Netherlands

Service Commun des Laboratoires Laboratoire de Montpellier Parc Euromédecine 205 rue de la Croix Verte 34196 Montpellier Cedex 5 France

U S Customs and Border Protection Laboratory 1100 Raymond Blvd Newark NJ 07102 USA

Zobrazit více v PubMed

Chang C, Jang-Liaw N, Lin Y et al. . Authenticating the use of dried seahorses in the traditional Chinese medicine market in Taiwan using molecular forensics. J Food Drug Anal 2013;21:310–6.

Lee SY, Ng WL, Mahat MN et al. . DNA barcoding of the endangered aquilaria (Thymelaeaceae) and its application in species authentication of agarwood products traded in the market. PLoS One 2016;11:e0154631. PubMed PMC

Milner-Gulland EJ, Bukreeva OM, Coulson T et al. . Conservation: reproductive collapse in saiga antelope harems. Nature 2003;422:135. PubMed

CITES www.cites.org. Accessed 12 September 2017.

Cheng X, Su X, Chen X et al. . Biological ingredient analysis of traditional Chinese medicine preparation based on high-throughput sequencing: the story for Liuwei Dihuang Wan. Sci Rep 2015;4:5147. PubMed PMC

Coghlan ML, Haile J, Houston J et al. . Deep sequencing of plant and animal DNA contained within traditional Chinese medicines reveals legality issues and health safety concerns. PLoS Genet 2012;8:e1002657. PubMed PMC

Coghlan ML, Maker G, Crighton E et al. . Combined DNA, toxicological and heavy metal analyses provides an auditing toolkit to improve pharmacovigilance of traditional Chinese medicine (TCM). Sci Rep 2015;5:17475. PubMed PMC

Ivanova NV, Kuzmina ML, Braukmann TWA et al. . Authentication of herbal supplements using next-generation sequencing. PLoS One 2016;11:e0156426. PubMed PMC

Taberlet P, Coissac E, Pompanon F et al. . Towards next-generation biodiversity assessment using DNA metabarcoding. Mol Ecol 2012;21:2045–50. PubMed

Staats M, Arulandhu AJ, Gravendeel B et al. . Advances in DNA metabarcoding for food and wildlife forensic species identification. Anal Bioanal Chem 2016;408:4615–30. PubMed PMC

Fahner NA, Shokralla S, Baird DJ et al. . Large-scale monitoring of plants through environmental DNA metabarcoding of soil: recovery, resolution, and annotation of four DNA markers. PLoS One 2016;11:e0157505. PubMed PMC

Arulandhu AJ, Staats M, Peelen T et al. . DNA metabarcoding of endangered plant and animal species in seized forensic samples. Genome 2015;188–9.

Taylor HR, Harris WE. An emergent science on the brink of irrelevance: a review of the past 8 years of DNA barcoding. Mol Ecol Resour 2012;12:377–88. PubMed

Little DP. A DNA mini-barcode for land plants. Mol Ecol Resour 2014;14:437–46. PubMed

Parveen I, Gafner S, Techen N et al. . DNA barcoding for the identification of botanicals in herbal medicine and dietary supplements: strengths and limitations. Planta Med 2016;82:1225–35. PubMed

RIKILT Wageningen University & Research www.decathlon-project.eu. Accessed 12 September 2017.

RIKILT Wageningen University & Research http://decathlon-fp7.citespipe-wur.surf-hosted.nl:8080/. Accessed 12 September 2017.

Chen R, Dong J, Cui X et al. . DNA based identification of medicinal materials in Chinese patent medicines. Sci Rep 2012;2:958. PubMed PMC

Scholtens I, Laurensse E, Molenaar B et al. . Practical experiences with an extended screening strategy for genetically modified organisms (GMOs) in real-life samples. J Agric Food Chem 2013;61:9097–109. PubMed

Murray MG, Thompson WF. Rapid isolation of high molecular weight plant DNA. Nucl Acids Res 1980;8:4321–6. PubMed PMC

Ivanova NV, Zemlak TS, Hanner RH et al. . Universal primer cocktails for fish DNA barcoding. Mol Ecol Notes 2007;7:544–8.

Edgar RC. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 2010;26:2460–1. PubMed

Altschul S. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucl Acids Res 1997;25:3389–402. PubMed PMC

Arulandhu AJ, Staats M, Hagelaar R. Development and validation of a multi-locus DNA metabarcoding method to identify endangered species in complex samples SOP. protocols.io 2017. http://dx.doi.org/10.17504/protocols.io.ixbcfin. PubMed DOI PMC

Hebert PDN, Cywinska A, Ball SL et al. . Biological identifications through DNA barcodes. Proc Royal Soc B Biol Sci 2003;270:313–21. PubMed PMC

Hollingsworth PM, Forrest LL, Spouge JL et al. . A DNA barcode for land plants. Proc Natl Acad Sci U S A 2009;106:12794–7. PubMed PMC

Liu S, Li Y, Lu J et al. . SOAP Barcode: revealing arthropod biodiversity through assembly of Illumina shotgun sequences of PCR amplicons. Methods Ecol Evol 2013;4:1142–50.

Gryson N. Effect of food processing on plant DNA degradation and PCR-based GMO analysis: a review. Anal Bioanal Chem 2010;396:2003–22. PubMed

Tang M, Hardman CJ, Ji Y et al. . High-throughput monitoring of wild bee diversity and abundance via mitogenomics. Methods Ecol Evol 2015;6:1034–43. PubMed PMC

CBOL www.barcodeofwildlife.org. Accessed 12 September 2017.

Galimberti A, De Mattia F, Losa A et al. . DNA barcoding as a new tool for food traceability. Food Res Int 2013;50:55–63.

Taberlet P, Coissac E, Hajibabaei M et al. . Environmental DNA. Mol Ecol 2012;21:1789–93. PubMed

Iyengar A. Forensic DNA analysis for animal protection and biodiversity conservation: a review. J Nat Conserv 2014;22:195–205.

16s Metagenomic Sequencing Library Preparation. Illumina document 15044223. https://support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf.

Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J 2011;17:10–12.

Schmieder R, Edwards R. Quality control and preprocessing of metagenomic datasets. Bioinformatics 2011;27:863–4. PubMed PMC

FASTX_Toolkit v. 0.0.14, http://hannonlab.cshl.edu/fastx_toolkit/.

ftp://ftp.ncbi.nlm.nih.gov/entrez/entrezdirect/.

UNEP-WCMC https://speciesplus.net. Accessed 12 September 2017.

Arulandhu AJ, Staats M, Hagelaar R et al. . Supplementary data for “Development and validation of a multi-locus DNA metabarcoding method to identify endangered species in complex samples.” GigaScience Database 2017. http://dx.doi.org/10.5524/100330. PubMed DOI PMC

Palumbi S, Martin A, Romano S et al. . The simple fool's guide to PCR, version 2.0. Privately published document compiled byPalumbi S. Honolulu, HI: Dept. of Zoology, University of Hawaii; 1991:96822.

Sarri C, Stamatis C, Sarafidou T et al. . A new set of 16S rRNA universal primers for identification of animal species. Food Control 2014;43:35–41.

Leray M, Yang JY, Meyer CP et al. . A new versatile primer set targeting a short fragment of the mitochondrial COI region for metabarcoding metazoan diversity: application for characterizing coral reef fish gut contents. Front Zool 2013;10:34. PubMed PMC

Geller J, Meyer C, Parker M et al. . Redesign of PCR primers for mitochondrial cytochrome c oxidase subunit I for marine invertebrates and application in all-taxa biotic surveys. Mol Ecol Resour 2013;13:851–61. PubMed

Parson W, Pegoraro K, Niederstätter H et al. . Species identification by means of the cytochrome b gene. Int J Legal Med 2000;114:23–28. PubMed

Fazekas AJ, Kuzmina ML, Newmaster SG et al. . DNA barcoding methods for land 39. Methods Mol Biol 2012;858:223–52. PubMed

Cuenoud P, Savolainen V, Chatrou LW et al. . Molecular phylogenetics of Caryophyllales based on nuclear 18S rDNA and plastid rbcL, atpB, and matK DNA sequences. Am J Botany 2002;89:132–44. PubMed

Levin RA, Wagner WL, Hoch PC et al. . Family-level relationships of Onagraceae based on chloroplast rbcL and ndhF data. Am J Botany 2003;90:107–15. PubMed

Kress WJ, Erickson DL, Shiu S. A two-locus global DNA barcode for land plants: the coding rbcL gene complements the non-coding trnH-psbA spacer region. PLoS One 2007;2:e508. PubMed PMC

Fazekas AJ, Burgess KS, Kesanakurti PR et al. . Multiple multilocus DNA barcodes from the plastid genome discriminate plant species equally well. PLoS One 2008;3:e2802. PubMed PMC

Taberlet P, Coissac E, Pompanon F et al. . Power and limitations of the chloroplast trnL (UAA) intron for plant DNA barcoding. Nucl Acids Res 2007;35:e14. PubMed PMC

Chen S, Yao H, Han J et al. . Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species. PLoS One 2010;5:e8613. PubMed PMC

Sang T, Crawford DJ, Stuessy TF. Chloroplast DNA phylogeny, reticulate evolution, and biogeography of Paeonia (Paeoniaceae). Am J Botany 1997;84:1120–36. PubMed

Tate JA, Simpson BB. Paraphyly of Tarasa (Malvaceae) and diverse origins of the polyploid species. Syst Botany 2003;28:723–37.

Manning J, Boatwright JS, Daru BH et al. . A molecular phylogeny and generic classification of Asphodelaceae subfamily Alooideae: a final resolution of the prickly issue of polyphyly in the alooids? Syst Botany 2014;39:55–74.

Protein Coding Low-Copy rpb2 and ef1-α Regions Are Viable Fungal Metabarcoding DNA Markers Which Can Supplement ITS for Better Accuracy

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