The challenges of working with eDNA
Top row – Ian Dickie, Rob Cruickshank, Jonathan Banks, Rob Smissen, David Orlovich, Sergio Morales, Gavin Lear, Andrew Dopheide, Syrie Hermans.
Front row – Jamie Wood, Stephane Boyer, Kim Handley.
Not pictured - Thomas Buckley, Robert Holdaway, Robin MacDiarmid, Charles Lee, Hannah Buckley.
Biodiversity assessment using environmental DNA
Given the importance of biological heritage for most aspects of our lives, surprisingly little is understood about the total diversity of life in New Zealand’s different ecosystems, and their vulnerability or reliance to pressures including land use and climate change. A first step in managing New Zealand’s biological heritage is simply to understand ‘what is out there’.
A barrier to achieving this is that monitoring the diversity of life in a sample or at a location is remarkably challenging. For this reason, existing biodiversity assessment protocols have typically focused on organisms that are easier to find and identify, for example our charismatic megafauna, and exclude the vast majority of organisms, such as fungi, invertebrates and microeukaryotes.
Forensic science has long revealed the potential to explore the diversity of life in a sample, not just from the DNA present within live cells, but from the skin, hair and bodily fluids of organisms inhabiting or transiting through an environment. It’s no longer necessary to sight an organism or an individual to confirm their presence the vicinity of a sampling location.
Recent advances in DNA sequencing technologies are revolutionising biodiversity monitoring from environmental DNA (eDNA). It is now possible to analyse the DNA molecules extracted from many hundreds of samples, simultaneously. This high throughput method is applicable to all organisms (i.e., archaea, bacteria, protists, fungi, animals and plants) simply by targeting different genetic markers.
eDNA analysis has potential as a universal tool for biodiversity and biosecurity assessments but according to Dr Gavin Lear there are some fundamental issues to be addressed.
“Despite the widespread appeal of this method, current approaches for sample collection, DNA extraction, storage, amplification and sequencing vary widely across laboratories and particularly among groups focusing on different taxa and sample media,” Gavin says.
“Comparisons of these data are therefore subject to multiple biases, many of which remain poorly quantified,” he adds.
A paper just published by Lear and his colleagues addresses this issue. The authors present a set of standard protocols for the identification of a broad range of taxa from the amplification of eDNA. The approaches outlined are designed to include coverage of both macro-organisms and microbial taxa and include specific protocols for the assessment of fungi, micro-eukaryotes, plants, animals, fish and prokaryotes.
The work is a game changer for researchers in the field. Wherever possible, methods are aligned with existing protocols, such as the Earth Microbiome Project recommendations to maximise opportunities for researchers from disparate groups to directly compare, or alternately combine data collected from their own study sites.
The standard set of methodologies provided, which includes in-depth guidelines for the assessment of multiple groups of taxa should further help to make eDNA analysis more accessible to less experienced users.
This ambitious effort could not be undertaken effectively by a single person, or even a single institution.
In February 2016, an open invitation workshop, facilitated by the The New Zealand Biological Heritage Challenge, ran to discuss and debate current and future research methods and opportunities in eDNA research. The workshop welcomed 55 participants from all of the country’s major universities and crown research institutes.
It marked the beginning of a coordinated effort to combine the skills of New Zealand’s scientific community to begin harmonising methods for the extraction, amplification, sequencing and storage of environmental DNA.
Gavin hopes this initiative may spur complementary efforts focused on an even greater variety of sample media, for example atmospheric DNA, as well as an even greater diversity of taxa, adding yet more layers of information regarding the appropriate use of eDNA in biodiversity monitoring.
The work of Gavin and his colleagues also highlights the role of National Challenges in New Zealand.
The New Zealand Biological Heritage Challenge was launched to coordinate efforts to better monitor, manage and protect New Zealand’s native biodiversity. This project is among one of many recent steps to harmonise frameworks for biological heritage monitoring across New Zealand’s diverse biological landscape.
Lear, G.; Dickie, I.; Banks, J.; Boyer, S.; Buckley, H.; Buckley, T.; Cruickshank, R.; Dopheide, A.; Handley, K.; Hermans, S., et al. 2017. Methods for the extraction, storage amplification and sequencing of environmental DNA (eDNA). New Zealand Journal of Ecology 42 (1). DOI: https://doi.org/10.20417/nzjecol.42.9
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.