A practical guide to DNA-based methods for biodiversity assessment

Kat Bruce; Rosetta Blackman; Sarah J. Bourlat; Ann Micaela Hellström; Judith Bakker; Iliana Bista; Kristine Bohmann; Agnès Bouchez; Rein Brys; Katie Clark; Vasco Elbrecht; Stefano Fazi; Vera Fonseca; Bernd Hänfling; Florian Leese; Elvira Mächler; Andrew R. Mahon; Kristian Meissner; Kristel Panksep; Jan Pawlowski; Paul Schmidt Yáñez; Mathew Seymour; Bettina Thalinger; Alice Valentini; Paul Woodcock; Michael Traugott; Valentin Vasselon; Kristy Deiner

doi:10.3897/ab.e68634

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https://doi.org/10.3897/ab.e68634 (02 Dec 2021)

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Acknowledgements
1. Introduction
- 1.1 The application of DNA-based methods to biomonitoring
- 1.2 Document structure
- 1.3 Sources and states of DNA
2. Capture, preservation and extraction of eDNA from water
- 2.1 Sampling strategy
- 2.2 Precipitation or filtration
- 2.3 Filtration method and filter type
- 2.4 On-site or off-site filtration
- 2.5 Volume of water
- 2.6 eDNA preservation strategy for filters
- 2.7 eDNA extraction from filters
3. Preservation and extraction of macroinvertebrate bulk samples
- 3.1 Preservation methods
- 3.2 Homogenisation methods
- 3.3 Non-destructive methods for macroinvertebrate bulk samples
- 3.4 Considerations for bulk DNA extraction
4. Sampling, preservation and extraction of benthic periphytic diatoms
- 4.1 Sampling benthic biofilms
- 4.2 Preservation methods
- 4.3 Considerations for DNA extraction from benthic biofilms
5. Sampling, preservation and extraction of soils and sediments
- 5.1 Collecting the sample
  - 5.1.1 Sample volume
  - 5.1.2 Sampling depth
- 5.2 Separation of organisms from soils and sediments
- 5.3 Preservation of sediment samples
- 5.4 DNA extraction from sediment samples
6. Measuring quality and quantity of extracted DNA
- 6.1 DNA quantification
- 6.2 Inhibition testing
- 6.3 Analytical controls used for DNA and eDNA analyses
7. Target species detection
- 7.1 Analysis methods for target species detection
  - 7.1.1 Conventional PCR (cPCR) assays
  - 7.1.2 Quantitative real-time PCR (qPCR)
  - 7.1.3 Droplet digital PCR (ddPCR) assays
- 7.2 Defining limits of detection (LOD) and quantification (LOQ)
- 7.3 Species specific primer design and validation
- 7.4 Quantitative interpretation of results
8. Metabarcoding to survey biological communities
- 8.1 Metabarcoding primer selection
  - 8.1.2 Amplicon length
- 8.2 Amplicon library preparation
  - 8.2.1 Types of library preparation
- 8.2.1.1 One-step PCR
- 8.2.1.2 two-step PCR
- 8.2.1.3 Ligation-based approach
  - 8.2.2 PCR replication
  - 8.2.3 Choice of polymerase
  - 8.3.1 Purification
  - 8.3.2 Quantification
  - 8.3.3 Amplicon diversity
9. Summary and advice
- 9.1 Factors influencing methodological choices
- 9.3 Concluding remarks
10. Glossary
11. References
12. Figure information and photo credits

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