How can we choose the ‘best’ DNA methods?
From 2017-2021, NatureMetrics cofounder Dr Kat Bruce led a working group on Field and Laboratory Methods within the EU COST Action programme DNAqua-Net . The aim was to develop consensus, best practice guidance and initial standards for the application of molecular monitoring in Europe and beyond.
This was no easy task. Collection and analysis of environmental samples for biodiversity assessment is a complex process incorporating many sequential steps. At each step, several choices can be made and there is little consistency in the published literature. This not only makes it difficult for researchers to select the best workflows for their research, but it also makes it hard for those commissioning DNA-based surveys (businesses, governments etc), to evaluate proposals that may vary substantially in their methods statements.
This methodological complexity is well illustrated by the aquatic eDNA metabarcoding workflow. Choices are made at every stage, including for:
- filter membrane materials and pore-sizes,
- sample preservation strategy,
- sampling strategy (how many samples, what volume, from how many points?),
- what kind of integrated positive controls to use,
- how to extract DNA and test for / remove inhibitors,
- how many PCR replicates to run,
- which primers to use with what type of library construction,
- whether to sequence replicates individually or pooled,
- and many more…
Moreover, methods are often non-independent of one another so a choice made at one point in the workflow may influence or constrain decisions that need to be made at other points. Research papers often compare individual methodological choices from the perspective of optimising results, but in applied settings and when delivering a full end-to-end workflow, other factors must also be taken into account. These include cost, complexity, logistical practicalities, health & safety, and contamination risk to name a few.
For instance, choice of sample preservation strategy may be driven by logistical considerations such as the ability to legally transport (or safely handle) flammable liquids, the availability of power for freezers, or the ability to maintain reliable temperature control during storage and transport, which may involve passing through customs checks in several countries. The best scientific choice is no good if it’s not logistically viable in the context of where the sampling will take place. The choice of preservation strategy will itself then influence the DNA extraction method – even just changing to a different liquid preservative solution demands adaptation of the early stages of the extraction process so that DNA is not lost.
This sounds daunting, and it certainly felt it when we embarked on the task of producing best-practice guidance. However, from our perspective at NatureMetrics, standardisation is something that we’ve worked hard to optimise from the start, allowing us to provide DNA services that are practical and applicable for our clients globally. In the early years of NatureMetrics, we based our methodological choices on our clients’ needs both in the field and for using the data. Our team have seen first-hand the importance of fully optimising each workflow, and ensuring the choice of methods considers the effect on the entire process, including the applicability of the final data. Our DNA workflows have now been very well tested in the field and are validated alongside other field methods to ensure the data is useful for a range of sectors from conservation to infrastructure and renewables.
Having completed the first edition of the practical guide, we have seen that our technology is based on a strong foundation of knowledge, and there was a high level of agreement on the core principles – even if the details vary and different users make different choices depending on their environmental, financial or logistical constraints.
What’s more, the approach is actually fairly robust to methodological variations. We’ve shown that the same results can be achieved by different workflows, even when different method choices have been made at multiple steps in the process.
Free download: A user guide for DNA monitoring
In The Practical Guide, we synthesise knowledge from across the scientific field, which was garnered through multiple international conferences and workshops involving the participation of hundreds of researchers, practitioners and end-users. The guide, whose 28 authors include many of the leading scientific experts in the field, considers four different types of samples (water, sediments, invertebrate collections and diatoms), and two primary analysis types (single species detection via methods such as qPCR and assessment of biological communities via metabarcoding). At each stage of the field and laboratory process the guide sets out the scientific consensus on best practice, as well as the choices that need to be made and the trade-offs they entail. In particular, the guide considers how the choices may be influenced by common practical constraints such as logistics, time and budget.
How did we begin the standardisation of eDNA and DNA methods?
Between 2016 and 2021, over 500 researchers collaborated within the DNAqua-Net international network, funded by the European Union’s European Cooperation in Science and Technology programme (COST), with the goal to develop and advance biodiversity assessment methods based on analysis of DNA obtained from the environment (e.g. river water) or from unsorted collections of organisms.
Available in an Advanced Book format, the guidelines will be updated as the technology continues to evolve.
Leaders of DNAqua-Net are Prof. Dr. Florian Leese of the University of Duisburg-Essen (Germany) and Dr. Agnès Bouchez of the French National Institute for Agriculture, Food, and Environment (INRAE). The core writing team for the present guide book involves Dr. Micaela Hellström (MIX Research AB, Sweden), Dr. Kat Bruce (NatureMetrics Ltd., UK), Dr. Rosetta Blackman (University of Zurich and EAWAG, Switzerland), Dr. Sarah Bourlat (LIB/Museum Koenig, Germany), and Prof. Kristy Deiner (ETH Zurich and SimplexDNA AG, Switzerland). You can read more about DNAquaNet at www.DNaqua.Net.