4.7 Article

An efficient and robust laboratory workflow and tetrapod database for larger scale environmental DNA studies

Journal

GIGASCIENCE
Volume 8, Issue 4, Pages -

Publisher

OXFORD UNIV PRESS
DOI: 10.1093/gigascience/giz029

Keywords

metabarcoding; invertebrate-derived -DNA; environmental DNA; leeches

Funding

  1. German Federal Ministry of Education and Research (BMBF) [FKZ: 01LN1301A]
  2. Leibniz-IZW
  3. MEME Erasmus Mundus Programme in Evolutionary Biology
  4. Groningen University Fund
  5. Marco Polo Fund from the University of Groningen
  6. National Natural Science Foundation of China [41661144002, 31670536, 31400470, 31500305]
  7. Key Research Program of Frontier Sciences, CAS [QYZDY-SSW-SMC024]
  8. Bureau of International Cooperation project [GJHZ1754]
  9. Strategic Priority Research Program of the Chinese Academy of Sciences [XDA20050202, XDB31000000]
  10. Ministry of Science and Technology of China [2012FY110800]
  11. State Key Laboratory of Genetic Resources and Evolution at the Kunming Institute of Zoology
  12. NERC [NE/M021696/1] Funding Source: UKRI

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Background: The use of environmental DNA for species detection via metabarcoding is growing rapidly. We present a co-designed lab workflow and bioinformatic pipeline to mitigate the 2 most important risks of environmental DNA use: sample contamination and taxonomic misassignment. These risks arise from the need for polymerase chain reaction (PCR) amplification to detect the trace amounts of DNA combined with the necessity of using short target regions due to DNA degradation. Findings: Our high-throughput workflow minimizes these risks via a 4-step strategy: (i) technical replication with 2 PCR replicates and 2 extraction replicates; (ii) using multi-markers (12S, 16S, CytB); (iii) a twin-tagging, 2-step PCR protocol; and (iv) use of the probabilistic taxonomic assignment method PROTAX, which can account for incomplete reference databases. Because annotation errors in the reference sequences can result in taxonomic misassignment, we supply a protocol for curating sequence datasets. For some taxonomic groups and some markers, curation resulted in >50% of sequences being deleted from public reference databases, owing to (i) limited overlap between our target amplicon and reference sequences, (ii) mislabelling of reference sequences, and (iii) redundancy. Finally, we provide a bioinformatic pipeline to process amplicons and conduct PROTAX assignment and tested it on an invertebrate-derived DNA dataset from 1,532 leeches from Sabah, Malaysia. Twin-tagging allowed us to detect and exclude sequences with non-matching tags. The smallest DNA fragment (16S) amplified most frequently for all samples but was less powerful for discriminating at species rank. Using a stringent and lax acceptance criterion we found 162 (stringent) and 190 (lax) vertebrate detections of 95 (stringent) and 109 (lax) leech samples. Conclusions: Our metabarcoding workflow should help research groups increase the robustness of their results and therefore facilitate wider use of environmental and invertebrate-derived DNA, which is turning into a valuable source of ecological and conservation information on tetrapods.

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