CERC eDNA Crew Blog - 2021
Designing eDNA assays
December 13, 2021
Last year our lab published a JoVE article and video on designing eDNA qPCR assays (Klymus et al., 2020). That is a lot of acronyms. JoVE is the Journal of Visualized Experiments. It produces short videos that depict a particular lab or field procedure so that others can follow the protocol. The journal covers a huge variety of scientific fields. For our article, we wrote an overview of the important considerations needed when designing qPCR (or quantitative PCR) assays for environmental DNA samples and in 2021 we were able to shoot the video portion of the article. (For an overview on eDNA and PCR see our post from 2020 CERC’s eDNA Crew Blog - 2020.)
We wrote the article because we found that designing the qPCR assay for eDNA sampling is an area that can be new for many researchers and even those with molecular experience may not be familiar with the particulars of qPCR. It is also the basis of eDNA research and thus proper assay design is vital in order to interpret the data.
Quantitative PCR or qPCR is a technique for measuring the amount of a specific target DNA sequence in a sample. By comparing the samples to a standard curve, we can measure the exact concentration of the target DNA in a sample. It is often used in medical and toxicology studies to measure changes in gene expression. It can be used as a diagnostic tool to detect human or wildlife pathogens (e.g. SARS-Cov-2 in humans, Bd in amphibians). Microbiologists also use qPCR to characterize microbial soil communities and bacterial function in the ecosystem. For eDNA, researchers use qPCR to quantify the amount of shed DNA from a target species in an effort to infer that species presence in the sampled area without physically capturing the organism. They usually design their qPCR assays to detect mitochondrial DNA sequences, because there is more mitochondrial sequence information available in the online databases compared to nuclear genome sequence, and mitochondrial DNA is present in many copies in each cell.
Quantitative PCR requires knowledge of the target sequence to design the assay, a standard solution of the target sequence at known concentration in copies/ul, a DNA polymerase enzyme that is stable when heated, and an instrument called a thermal cycler that drives the PCR and measures the fluorescence of the reaction during each thermal cycle.
A key goal is to make sure the assay only amplifies the target molecule, which in the case of eDNA is the target species DNA. If you have closely related species in the area being sampled, the primers might also amplify those species. This can make it difficult and not always possible to design truly species-specific assays. For example, we were unable to design qPCR assays to distinguish shovelnose sturgeon from pallid sturgeon because their mitochondrial DNA sequences are so similar.
Another important aspect of designing qPCR assays for eDNA is the testing, we follow three steps in testing the assays: 1)in silico – test the designed assay against a public or curated sequence database of co-occurring and closely related species, 2) in vitro – test the assay in the lab with DNA extractions from co-occurring and closely related species and 3) in situ – test the assay with eDNA samples from field sites in which the presence or absence of the target species is known. Thalinger et al. (2021) also provide a good overview of these steps. For another example of a freshwater mussel assay design following these steps, see Currier et al. (2018).
Due to covid restrictions the video production was delayed until this past spring. Working to produce a video was an interesting process. We first had to go through several iterations of the script and make detailed notes on exactly what we wanted the videographer or video production team to show, whether it was an animated or still picture, a computer screen recording, or a video of us doing part of the procedure. We had to avoid any extra items or actions in the shots so that the subject would be clear in each segment. The day of the video shoot, our videographer had us do several takes of each part so that the video production team would have enough material when putting the full video together. This was our only chance to get the video portion. It was also cold and windy, but we needed to do the interview portions outside, so I was not looking my happiest in the interview….I really dislike the cold weather. We also had to do several takes because cars kept passing by. Overall, it was an interesting experience and I have a lot of appreciation for people who do video production, because it is really more than just taking one long video. It took all day to generate the footage for our 9-minute video. We hope that the article and video can be of use to the eDNA community and especially those researchers that are just getting into the field.
Klymus, K.E., Ruiz Ramos, D.V., Thompson, N.L., and Richter, C.A., 2020, Development and testing of species-specific quantitative PCR assays for environmental DNA applications: JoVE, no. 165, p. e61825.
Thalinger, B., Deiner, K., Harper, L.R., Rees, H.C., Blackman, R.C., Sint, D., Traugott, M., Goldberg, C.S., and Bruce, K., 2021, A validation scale to determine the readiness of environmental DNA assays for routine species monitoring: Environmental DNA, v. 3, no. 4, p. 823-836.
Currier, C.A., Morris, T.J., Wilson, C.C., and Freeland, J.R., 2018, Validation of environmental DNA (eDNA) as a detection tool for at-risk freshwater pearly mussel species (Bivalvia: Unionidae): Aquatic Conservation: Marine and Freshwater Ecosystems, v. 28, no. 3, p. 545-558.
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