By Christina Nardini (SARA intern 2024, Masters by Placement, James Cook University)
Recently, as part of my internship with SARA, I had the chance to visit the TropWATER eDNA lab in Townsville. This lab’s purpose is to process water and soil samples from the field to detect the presence of species. It was an exciting opportunity to witness firsthand how eDNA (environmental DNA) is extracted and analyzed, and how crucial this work is to conservation efforts.
A Non-Contamination Lab for a Clean Start
As I entered the non-contamination lab, the first step in the eDNA process, I noticed how everything was carefully controlled to prevent contamination. Lab staff were required to wear shoe coverings, gloves, and lab coats. With every surface and tool sterilized with bleach or UV light, it was clear how important it is to eliminate contamination and ensure no outside DNA interfered with the samples. This level of precaution is necessary to maintain the integrity of the results.
Step-by-Step: The eDNA Analysis Process The lab follows a five-step process to analyze eDNA, each with meticulous attention to detail:
1. Sampling: Water samples are collected in the field and transported to the TropWATER lab in sealed jars. These samples can be stored for up to three months, thanks to a preservative solution called Longmire’s Buffer. Multiple samples from different sites are sent in, with replicates used to ensure accuracy. When it comes to searching for sawfish DNA, SARA’s samples arrive as filter paper stored in Longmire Solution, as we sample large quantities of water by filtering it through the filter paper.
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Samples arrive to the lab in sample jars with lids taped shut and in bags to prevent any loss of samples (Figure A). These samples can be stored for up to 3 months thanks to the Longmire’s Buffer (Figure B). When setting up test tubes for the DNA extraction process, 25 test tubes are needed per sample/yellow-lidded jar (Figure C). Of these, 5 are used per replicate, where one is used as a control group and 4 as treatment groups.
2. DNA Extraction: This is where the real work begins. In the non-contamination lab, researchers first prepare the samples by carefully cleaning the jars and labelling all equipment. Each sample is divided into test tubes, with some used for treatment and others as controls. The samples are mixed with glycogen, NaCl, and isopropanol before being left overnight in a fridge. The next day, the samples are spun in a centrifuge, separating the DNA for extraction.
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Lab tech Emma seen adding glycogen to each test tube (Figure D.) Figure E shows one of the many centrifuges in the non-contamination lab.
3. Inhibition Tests: After extracting DNA, inhibition tests are conducted to ensure there are no substances in the sample that might interfere with the PCR process.
4. qPCR (Quantitative Polymerase Chain Reaction): In this step, scientists use qPCR to detect the DNA of specific species, like sawfish, in the sample. The lab was color-coded by task—blue for the DNA extraction lab, and yellow and green for different sides of the pre-PCR labs. It was essential to maintain strict lab separation to avoid cross-contamination. The team even scheduled shifts so that those working in one area of the lab couldn’t potentially contaminate another.
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Different sides of the lab are used for different steps in the qPCR process. These are colour-coded by step, blue for the DNA extraction lab (Figure F), yellow and green for different sides of the pre-PCR labs (Figures G and H).
5. Data Analysis: Once the DNA is amplified and species-specific markers are detected, the data is analyzed to determine whether sawfish DNA is present in the samples. This critical information is then sent back to those who sent in the samples, like Sharks and Rays Australia. This eDNA data is then used to write reports, inform conservation efforts, and aid in research.
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Figure I shows a qPCR machine used to detect DNA in samples. The amplification of the DNA is shown in Figure J, where the data is output for analysis and distribution.
Why It Matters
The analysis of eDNA is a new method that provides insights into species distributions and environments. This lab gives organizations and researchers the ability to use non-invasive sampling techniques to study species and environments. With TropWATER’s help, Sharks And Rays Australia can gather data on sawfish distribution quicker and easier than with previous methods.
My visit to the TropWATER eDNA lab gave me a new appreciation for the precision and dedication that goes into this work. From the painstaking steps to avoid contamination to the advanced technology used in DNA detection, every part of the process is geared toward one goal: understanding and conserving our natural world.
SARA’s eDNA work is supported by various funding bodies and grants, including the Sea World Research and Rescue Foundation Inc., the Reef Trust Partnership and Great Barrier Reef Foundation, and a Community Sustainability Action Grant from the Queensland Government.