New Zealand Journal of Ecology (2018) 42(1): 10

Methods for the extraction, storage, amplification and sequencing of DNA from environmental samples

Review Article
Gavin Lear 1*
Ian Dickie 2
Jonathan Banks 3
Stephane Boyer 4
Hannah L. Buckley 5
Thomas R. Buckley 1,6
Rob Cruickshank 7
Andrew Dopheide 6
Kim M. Handley 1
Syrie Hermans 1
Janine Kamke 1
Charles K. Lee 8
Robin MacDiarmid 9
Sergio E. Morales 10
David A. Orlovich 11
Rob Smissen 12
Jamie Wood 12
Robert Holdaway 12
  1. School of Biological Sciences, University of Auckland, 3a Symonds Street, Auckland 1010, New Zealand
  2. School of Biological Sciences, University of Canterbury, Christchurch 8140, New Zealand
  3. The Cawthron Institute, Nelson 7010, New Zealand
  4. Insect Biology Research Institute (IRBI) – UMR 7261 CNRS / Université François-Rabelais de Tours, Parc Grandmont, 37200 Tours, France
  5. School of Science, Auckland University of Technology, Auckland 1010, New Zealand
  6. Landcare Research, Auckland 1142, New Zealand
  7. Department of Ecology, Lincoln University, Lincoln 7647, New Zealand
  8. School of Sciences, The University of Waikato, Hamilton 3240, New Zealand
  9. Plant and Food Research, Auckland 1025, New Zealand
  10. Microbiology and Immunology, University of Otago, Dunedin 9054, New Zealand
  11. Department of Botany, University of Otago, Dunedin 9054, New Zealand
  12. Landcare Research, Lincoln 7640, New Zealand
*  Corresponding author

Advances in the sequencing of DNA extracted from media such as soil and water offer huge opportunities for biodiversity monitoring and assessment, particularly where the collection or identification of whole organisms is impractical. However, there are myriad methods for the extraction, storage, amplification and sequencing of DNA from environmental samples. To help overcome potential biases that may impede the effective comparison of biodiversity data collected by different researchers, we propose a standardised set of procedures for use on different taxa and sample media, largely based on recent trends in their use. Our recommendations describe important steps for sample pre-processing and include the use of (a) Qiagen DNeasy PowerSoil® and PowerMax® kits for extraction of DNA from soil, sediment, faeces and leaf litter; (b) DNeasy PowerSoil® for extraction of DNA from plant tissue; (c) DNeasy Blood and Tissue kits for extraction of DNA from animal tissue; (d) DNeasy Blood and Tissue kits for extraction of DNA from macroorganisms in water and ice; and (e) DNeasy PowerWater® kits for extraction of DNA from microorganisms in water and ice. Based on key parameters, including the specificity and inclusivity of the primers for the target sequence, we recommend the use of the following primer pairs to amplify DNA for analysis by Illumina MiSeq DNA sequencing: (a) 515f and 806RB to target bacterial 16S rRNA genes (including regions V3 and V4); (b) #3 and #5RC to target eukaryote 18S rRNA genes (including regions V7 and V8); (c) #3 and #5RC are also recommended for the routine analysis of protist community DNA; (d) ITS6F and ITS7R to target the chromistan ITS1 internal transcribed spacer region; (e) S2F and S3R to target the ITS2 internal transcribed spacer in terrestrial plants; (f) fITS7 or gITS7, and ITS4 to target the fungal ITS2 region; (g) NS31 and AML2 to target glomeromycota 18S rRNA genes; and (h) mICOIintF and jgHCO2198 to target cytochrome c oxidase subunit I (COI) genes in animals. More research is currently required to confirm primers suitable for the selective amplification of DNA from specific vertebrate taxa such as fish. Combined, these recommendations represent a framework for efficient, comprehensive and robust DNA-based investigations of biodiversity, applicable to most taxa and ecosystems. The adoption of standardised protocols for biodiversity assessment and monitoring using DNA extracted from environmental samples will enable more informative comparisons among datasets, generating significant benefits for ecological science and biosecurity applications.