Every eDNA survey needs to have clearly laid out where, when, and how the samples will be collected. These decisions are based on a number of factors including the research objective, the project budget, and the logistics of field collection.
Stage outcomes
The main outcome from this stage is an improved understanding of the major workflows associated with eDNA sampling and how these can be used to achieve your objectives. You will also be able to identify key questions and considerations to address when designing a survey.
Establishing Objectives
What is your overall objective?
Many of the decisions on the survey design will depend on the specific objectives and targets of the survey.
For example, a general biodiversity survey might maximize the number of locations and habitats sampled to increase the diversity of species detected, while a survey targeting a single species might focus on a particular habitat, season, or depth. Similarly, a survey aimed at detecting a rare species may require larger samples or greater replication than a survey targeting more abundant taxa.
How does my research objective affect my eDNA workflow?
Your research objective will also largely determine what sample processing approach you will need to take, as well as what information you can infer from your outputs. When employing eDNA in biodiversity science, two major approaches are used.
DNA metabarcoding is useful if you want to get a picture of whole communities of fish or invertebrates or other groups of organisms in the environment. This process sequences the DNA in your sample to identify multiple species at once, across hundreds of samples simultaneously, to rapidly assess biodiversity across large geographic or temporal scales.
If you would like to understand more about the limitations of eDNA these approaches, please consider reading Using eDNA for Environmental Management.
Survey Design Considerations
What depth to sample?
An important consideration is deciding where in the water column to collect the sample. This decision will be based on the water depth at each site, the species being targeted, and the equipment that you have available.
It is often desirable to collect water samples at multiple depths from each location to better measure the total biodiversity and understand how it changes with depth. Sampling at multiple depths can also be helpful if trying to detect a rare species.
How far apart should locations be?
The distance between sampling locations depends on the research question and the geography of the study area. For example if there are different micro-habitats of interest (kelp forests, seagrass beds, rocky reefs) then eDNA can be a good tool for measuring the unique diversity of each habitat, even if they are close together. However, a broader scale census of biodiversity is also possible with samples placed farther apart. Other factors like the depth, the currents, and freshwater inputs can all affect the decision about how close to sample.
Pilot studies can be helpful for fine-tuning this aspect of survey design.
Should samples be collected at multiple time points?
If you are interested in changes through time or seasonal patterns in biodiversity, then identifying a set number of locations that can be repeatedly sampled can be an excellent way to monitor ecosystem changes. The frequency of sampling (i.e. monthly, seasonally, annually) will depend on the research question, timeline of the project, and budget.
How many replicates should I collect?
Sample replicates, where multiple samples are collected at the same time and place, are helpful for evaluating false positive and false negative detections and can improve the ability to detect rare species. We suggest anywhere from 3-5 replicate samples can help to ensure a robust eDNA sample design.
How many field blanks do I need?
Field blanks, or controls, are essential for identifying contamination during an eDNA survey. One approach is to process sterile or deionized water using the same methods (and in parallel with) the actual eDNA samples.
For example, sampling from a boat that has been used for fishing may introduce DNA contamination into the samples, however this contamination will be detected in the field blank allowing you to make adjustments to the interpretation of the signal in the real eDNA samples.
The number of field blanks depends on how many samples are being collected. Generally incorporating 1-3 field blanks per day is a good method to minimize errors in the eDNA samples.
What volume should I collect for each sample?
Each sample used in the survey should target the same volume of seawater. Samples with larger volumes will increase the likelihood that rare species are detected, however there is a trade-off as larger volumes are more time-consuming to process and can clog the filters if the water is turbid. We recommend that samples in the range of 1-2L are a good starting point for most surveys targeting marine invertebrates and vertebrates.
Resources
The following resources are associated with the Planning stage of the eDNA biodiversity monitoring method.
Next stage
Once you are more familiar with your research objectives and associated key questions and considerations, you will be ready to move on to the Collection stage.
