eDNA Best Practices- Resource Manager’s eDNA Toolbox Active

Best Practices and Data Standards

• Develop a Communication Plan
• Study Design and Pilot Study
• Assay Validation
• Sample Collection
• Laboratory Analysis
• Data Analysis and Interpretation- Communication

Study Design and Pilot Study

Before starting an eDNA study, assemble an interdisciplinary team to decide on goals, select proper methodologies, develop study design, and talk about the communications plan. Certain steps are recommended before you begin fieldwork.^[7]

Study Design

• Engage with lead management authorities, partners, or other interested parties to decide on a communication plan and the definition of a positive detection.
• Use probe-based quantitative PCR when targeting a single species or genus.
• When targeting communities or unknown species, use eDNA metabarcoding with high-throughput sequencing.
• You must validate eDNA assays in silico, in vitro, and in situ.
• It’s recommended to run a pilot study to assess detection probabilities with given sampling methods.
• Test extraction and analysis protocols.
• Consider a sampling scheme where multiple samples are collected across time. Resampling of the same regions can provide support and inform data interpretations. 
• Assess any potential alternative sources of eDNA.

Pilot Studies

Pilot studies are essential for eDNA studies due to their ability to identify variables that will affect eDNA detection. Since every study is different, protocols must be tested to detect any issues with the sample design or assay validation. It's important to identify study objectives and lay out the thought process by asking questions such as:

• What are the best locations to collect your samples?
• What is the best season to sample?  How often do you re-sample the same region to inform data interpretations? 
• How often do you sample?
• How many samples do you have to collect to detect a population of a given size you want to find? How small of a population can you detect with a given amount of effort you are willing to put in?
• What is the optimal filter material, filter pore size, and filtered volume?
• What is the optimal sample collection method for your water quality parameters? These parameters may include turbidity, organic content, salt content, pH, inorganic particle size distribution, etc.

Validation, Quality Assurance, and Quality Control

Data generated from eDNA studies must be reliable and follow high-quality standards.^[10] Assay development, testing, and validation are all crucial steps in eDNA studies.

Assay validation

Assay validation is the process of proving that the performance of an assay is sensitive and specific. Validation is one of the most significant steps in eDNA studies. When developing an assay, there are three required steps:

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In silico- assay validation needs to be conducted through comparisons with reference sequences for the target species and species co-occurring in the sampled habitat.

In vitro- The assay is then tested for specificity and sensitivity in the lab using quantitative PCR.

In situ, habitat validation- involves applying the assay to eDNA samples from environments where your target species is present as well as environments where it’s absent.^[7] The goal of in situ testing is to confirm positive marker amplification in eDNA samples from sites where the target species is known to be present. Another important step is confirming that eDNA samples won’t positively amplify in sites where the target species is presumed absent.^[21]  The assay must use target eDNA positive water or sediment samples that you have obtained from the geographic region of your study.

A 5-level validation scale was developed by Thalinger et al. 2021 to provide guidance on assay validation. However, data collected using assays that don’t meet this high bar of complete validation described by Thalinger et al. should not be automatically dismissed.

Quality Assurance and Quality Control

Quality assurance and quality control standards that are chosen for your project should be agreed upon by all parties involved.^[13] For qPCR standards, we recommend following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. MIQE is a set of guidelines that define the minimum information that is needed when you are conducting qPCR experiments.^[3] These guidelines help improve the quality of qPCR experiments in eDNA leading to more reliable results. There is also recommended information to be included in Goldberg et al. table 1 and table 2.^[7] 

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Limit of Detection and Limit of Quantification

For targeted studies, you should define the limit of detection (LOD) and limit of quantification (LOQ).^[9] 

• Effective Limit of detection (LOD) – the lowest concentration of DNA that can reliably be detected (such as with 95% confidence) given how many replicates each sample was analyzed.
• Limit of quantification (LOQ) – the lowest concentration with a coefficient of variation below a given value, usually ranging from 25% to 35%.^[10] Others have defined LOQ as the lowest concentration where at least 90% of the technical replicates can be detected.^[8][12]

These limits are found by running multiple replicate standard curves of an assay and then modeling detection rates and coefficients of variation (CV). 

When reporting LOD and LOQ values, we encourage you to also describe the laboratory and mathematical methods used including any specified criteria applied.

For more information on LOD and LOQ, see Lesperance et al. 2021.

Sample Collection

The steps below are recommended once fieldwork begins.^[7]

• Use negative controls at each phase of the process.
• Strict decontamination protocols must be set up for all supplies and equipment to reduce contamination possibility.
• Multiple samples must be collected from each site to improve detection probabilities and estimate false negatives likelihoods.
• Samples should be preserved immediately after collection to prevent degradation. 
• Samples should be clearly labeled to make them easily identifiable. Consider the use of barcodes or QR codes for sample labeling. 
• Metadata must be collected for every eDNA sample during field sampling. This can include sample collection personnel, sample geographic location, sample date, water temperature, and weather conditions to name a few.

A site refers to a specific, physical area where a sample has been collected from. 

Replicates are important for eDNA usage because they increase the probability of detection. Field replicates are separate samples collected as close as possible in time and space. Technical replicates are PCR replicates where the same DNA sample is tested in separate reactions. Running multiple replicates per sample improves the probability of detecting target DNA.^[1][5]

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Laboratory Analysis

The critical considerations described by Goldberg et al. are widely accepted as required practices for obtaining valid results.^[7] 

• Process samples in a dedicated clean laboratory (separate from PCR products) with restricted access, regular decontamination (bleach, UV) and use filtered pipette tips.
• Use negative controls at each phase of the process.
• Use technical replicates to assess repeatability of the data and internal positive controls to test for inhibition.

eDNA Concentration

Methods of eDNA concentration involve filtration, centrifugation, and isopropanol or ethanol precipitation.^[15]

• Filtration is suitable for a variety of water volumes, often used with 250 mL -45L of water.
• Centrifugation and precipitation are used for collecting small volumes (<250 mL).

Extraction Methods

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  For eDNA detection, most studies have used commercial extraction kits while other studies have used salt DNA extraction methods, cetyl trimethylammonium bromide (CTAB), or phenol-chloroform-isoamyl alcohol (PCI).^[15]

• The choice of which commercial extraction kit works best will depend on your specific habitat, species, budget, and other preferences.

Inhibition

Substances, such as humic acids, that are present in the eDNA sample could negatively affect the reliability of your eDNA analysis. Inhibition can prevent amplification and can increase the potential for false negative detections. You must test for PCR inhibition before you can trust your quantification or any negative test results. To test for inhibition, an internal positive control (IPC) must be added to your sample. There are commercial inhibitor removal kits available to help aid you and your projects.^[1]

Data Analysis and Interpretation

Finally, we recommend certain guidelines for reporting eDNA results and challenges.^[6][7]

• Acknowledge challenges inferring across space and time: study designs should understand factors such as the processes that move and degrade eDNA in the environment.
• Resample the same regions to provide additional support for the data/inform data interpretations.
• Confounding sources of eDNA – A species’ eDNA presence does not necessarily mean that a species is present in the environment. It’s possible that the eDNA was introduced to the environment by other means such as sewage or feces. Interpretations of DNA degradation and movement (fate and transport, respectively) will remain an area of interest for further research. It’s important to:
  • Ground truth data when possible.
  • Repeat sampling to confirm results.
  • Conduct a thorough assessment of background DNA concentrations.
  • Make sure sampling practices facilitate interpretation. For example, sample up stream until the signal is no longer detected to find the most upstream point where eDNA has been deposited.