CERC eDNA Crew Blog -2023
CERC eDNA Crew Blog - 2023
A Day in the Life in an eDNA Lab
The USGS Columbia Environmental Research Center (CERC) is home to an aquatic species environmental DNA (eDNA) laboratory that plays a significant role in endangered and invasive species monitoring. Environmental DNA is an identification method that is used to detect the presence of species in an environment, often aquatic systems, using DNA left behind by the organisms present in the water. Follow our lab technicians through a day in the lab to gain an understanding of how this cutting-edge technology works.
Anton’s Day: Water Sampling from the Grass Carp Pond
To kick off an experiment we first must collect samples from a target area. For this experiment we wanted to compare the amount of grass carp DNA collected using grab samples versus glass fiber filter samples.
As I start my day, I am getting ready for collecting water samples from a pond that contains grass carp. My goal is to collect grab samples of 50 milliliters of water, and filter 1 liter samples of water onto glass fiber filters without any outside material that could possibly contaminate the sample. I am using a blank to ensure that my sampling was done properly. If the blanks come up as positive the samples associated with the positive blanks would become invalid. Therefore, I need to be precise and careful while collecting the samples so that we can be confident in our results. I am bringing 50 mL tubes and, the pump and the filtering system to the pond. Typically, I take a grab sample first. I dip the tubes into water, collecting 50 mL in each.
The next step is to filter water with the glass fiber filters. I set up the pump system by attaching four filters to the sampling pole which has hoses leading to the pump system and then to a waste bucket for measuring the volume of water being collected. I place the collection hoses for the filters into the water and turn the pump on, as the water passes through the glass fiber filter the DNA and the cells that are in water will stick to the filter. I am letting the pump system run until we have collected at least one liter of water per filter. After I have collected enough water, I turn off the pump and detach the filters. I use gloves to open the filtering cases and collect the glass fiber filter with forceps, folding it to place it into a 2mL tube that already contains ethanol for preservation. I will repeat these two sampling processes until I have taken samples from all the collection sites around the pond.
The last part of the water sampling is to take all my samples to the sample processing lab. I place the glass fiber filter tubes into the freezer. Then the grab samples are placed into a centrifuge; this is done to separate the dense DNA, epithelial cells, and waste material out of the water and into a tiny pellet at the bottom of the tube. The centrifuge spins the tube fast enough to fix the pellet at the bottom so we can pour the water from the tube, leaving only the cells at the bottom. My last step is to add buffer and vortex the tubes to resuspend the biological material, and then place them in a freezer until I need to extract the DNA at later date to complete our experiment.
Megan’s Day: Sample processing, digestion, and extraction
I start my day by preparing collected samples for DNA extraction. I add a storage lysis buffer to the filter samples collected previously. The buffer will stabilize the DNA throughout processing to prevent degradation. These samples were collected a few days ago as part of a freshwater mussel species field monitoring study whose results will hopefully give us an indication of where the mussels are living in the river.
I use a technique called bead beating, which allows the collected cells/DNA to detach from the filter and be fully resuspended in the buffer. At this point, samples can be refrozen, but since I have time, I will continue processing them through digestion and extraction. I add a small amount of an enzyme called Proteinase K to each sample that will break open the cells and expose the DNA contained within. While those are digesting in a preheated water bath, I take the time to set up the extraction robots which are an automated system that will separate the newly free DNA from the rest of the cellular material in the sample, giving us a clean DNA sample ready to be amplified for analysis. I make sure to include an extraction blank with every set that I extract to check for contamination in this step of the workflow. After the extraction is complete, I move these samples to our clean lab so I can run a quantitative polymerase chain reaction (qPCR) on them later this week.
Emile’s Day: qPCR
Today I am working primarily on running qPCR reactions to quantify the amount of Mucket (Actinonaias ligamentina) DNA in previously collected samples. Mucket mussels are not federally endangered, but we use them as a comparison species when investigating population levels of endangered mussels. I extracted these samples earlier this week, so the DNA is stored in CDT buffer and is ready to be run on a real-time PCR thermocycler. I start setting up this qPCR by making mastermix, which consists of buffers, Taq polymerase enzyme, and a set of carefully crafted primers and probes specific for a section of the species’ mitochondrial genome that will bind with the DNA. I also make my standards; these are a set of known DNA concentrations that can be compared to the values obtained from my samples after the PCR reaction is completed. I then plate the mastermix, standards, and samples by adding precise amounts of each to the wells on a 96-well plate, which allows for the amplification of up to 24 samples in triplicate reactions.
Once I am done plating, I seal the plate and put it in a centrifuge to pull down any extra drops of liquid on the side of wells that may change the results of the qPCR testing. I can now put the plate in the thermocycler; this machine heats and cools the plate, which allows for the primers to bind the DNA at a selected location and for the polymerase to extend and amplify this selected region. The probe is degraded by the polymerase as it binds to the new strands of target DNA causing it to release a fluorescent label, a signal that can be picked up by the machine and read as an indication of how much DNA is in the reaction. The amount of DNA in the reaction for each sample is then compared to the known standards, which I can ultimately use to determine the amount of target DNA that was originally in the sample.
Collecting water samples, extracting DNA from these samples, and quantifying that DNA in a qPCR are all integral parts of our daily activities in the CERC eDNA Lab. Results from our lab after this workflow can provide valuable knowledge about of the presence of endangered and invasive species in a variety of aquatic habitats. This knowledge is invaluable in informing further conservation efforts conducted by our collaborators.
