Environmental DNA, or eDNA, is defined in the traditional sense as the mixture of genetic material released from an organism into its environment. Taking advantage of the fact that organisms can shed DNA via skin, hair, scales, feces, or bodily fluids as they move through their aquatic or terrestrial environment (1), eDNA analysis has revolutionized how field biologists can detect and monitor endangered species.
As in forensic science, researchers are able to track a trail of environmental DNA. This means that a particular species of fish migrating through a river, or a virus that has passed through wastewater, can now be detected even though they are no longer physically present in that sample. Diluted as such, eDNA is therefore often present only in vanishingly small quantities. It is only in the last decade that novel molecular techniques for detection and analysis, coupled with the ability to filter the sample on-site, have been available.
Without a sample filtration/concentration step, would eDNA be lost in the environment?
In general, eDNA analysis involves the following steps: capture, preservation, extraction, amplification, and sequencing. Efficiency at each stage has a knock-on effect on the output of subsequent steps; hence, the initial steps of capture, preservation, and extraction are especially important as they directly impact the quantity and quality of DNA available for amplification and sequencing.
Because eDNA detection often relies on detecting ultra-low sample concentration of highly degraded DNA, filtration is typically preferred as it enables the collection of eDNA from large volumes of water or other media. Filtration (the passage of water samples through a filter to trap the DNA) is preferred over precipitation (using ethanol to precipitate nucleic acids in the sample) as the critical capture method step. A study by Hinlo et al. (2) investigated filtering 250 mL samples through a 47 mm, 0.8 μm membrane filter using a filter funnel manifold and a pump (Pall™ Life Sciences products) in a set-up similar to Figure 1. They compared it to a precipitation method and found that the method involving filtration yielded high quantities of DNA. This is consistent with other studies that also showed that filtration recovered more eDNA from water samples (3, 4).
Fig 1. MicroFunnel Filter Funnel Unit and Manifold (Pall™ Life Sciences products).
Different filtration devices can be used in the capture step of eDNA analysis. The choice of filtration device depends on both workflow handling requirements and the initial sample volume. Commonly, sample volumes ranging from 15 mL to 5 L are used for analysis. Cytiva supplies a range of filtration devices, including syringe filters, MicroFunnel™ filtration units, and larger filter capsule devices. We also supply membrane discs and filter holders
Our pre-sterilized MicroFunnel™ filtration units are available in 100 mL and 300 mL formats. MicroFunnel filter funnels come ready-to-use, fully assembled, with both filter membrane and funnel. They are available with a range of membrane chemistries including GN-6 Metricel™ (mixed cellulose esters) membrane, Supor™ (hydrophilic polyethersulfone [PES]) membrane and Metricel™ Black PES membrane.
We supply a wide range of filter capsule devices with different effective filtration areas (EFA) to satisfy the throughput of different sample volumes with varying turbidity. Our capsules feature a range of membrane chemistries including Supor™ membrane.
Our filtration devices are available with different pore sizes including 0.2 μm, 0.45 μm and 0.8 μm. While there is no consensus on the exact pore size that should be used, a pore size of 0.45 μm appears to be the most commonly used, according to the published literature.
Time of filtration and storage
DNA can degrade in the environment, and the rate of eDNA degradation increases with higher temperatures and exposure to UV light. Therefore, it can be important to reduce the time between sampling and filtering for optimal eDNA recovery (5).
For on-site sampling researchers may use an electrical vacuum pump connected to a filter holder manifold, which allows researchers to combine benefits of both on-site and laboratory filtering by ensuring optimally fast and sterile filtering conditions are observed during sample collection.
An electrical pump and manifold were used in the Nature Research report by Majaneva et al. (6), where they examined how eDNA filtration techniques affect recovered biodiversity. In their comparative study, the researchers chose to filter the samples at the sampling site and perform immediate filter preservation to minimize time for eDNA decay in the samples.
Fig 2. Sentino™ Pump and MicroFunnel™ Filter Funnel Units (Pall™ Life Sciences products).
The Sentino pump is well-suited for use in the field due to its small footprint and battery operation. Its peristaltic flow design means the sample is pulled through the filter and fluid path, eliminating the need for a vacuum source. It also ensures the fluid flows uniformly in one direction without the potential for back-up and contamination of the sample. The Sentino pump has been designed to be fully compatible with filtration devices such as the MicroFunnel filter funnels and our reusable magnetic filter funnels.
The use of eDNA analysis is rapidly evolving. Once a tool used predominately within the field of ecology, its breadth of applications has recently exploded and will no doubt continue to grow rapidly in the foreseeable future. In fact, most recently, researchers have begun to repurpose eDNA techniques to detect eRNA in human wastewater. These genetic tools have been employed to better understand and monitor the SARS-CoV-2 virus driving the global COVID-19 human pandemic. SARS-CoV-2 viral shedding in fecal matter takes place in infected individuals, regardless of whether that individual is symptomatic, and its detection in human wastewater can provide data for the spread of COVID-19 in communities.
To learn more about how additional Cytiva products have been integrated into eDNA workflows by the scientific community, please read the Scientific Brief: The Importance of Filtration in the eDNA World
References
- Seymour M. Rapid progression and future of environmental DNA research. Commun Biol. 2019;2:80. Published 2019 Feb 27. doi:1.1038/s42003-019-0330-9
- Hinlo R, Gleeson D, Lintermans M, Furlan E. Methods to maximise recovery of environmental DNA from water samples. PLoS ONE. 2017;12(6):e0179251. https://doi.org/10.1371/journal.pone.0179251
- Deiner K, Walser J-C, Mächler E, Altermatt F. Choice of capture and extraction methods affect detection of freshwater biodiversity from environmental DNA. Biolog Conserv. 2015;183(0):53–63. https://doi.org/10.1016/j.biocon.2014.11.018
- Piggott MP. Evaluating the effects of laboratory protocols on eDNA detection probability for an endangered freshwater fish. Ecol Evol. 2016;6(9):2739–50. Published 2016 Mar 17. doi:10.1002/ece3.2083
- Turner CR, Barnes MA, Xu CCY, Jones SE, Lerde CL, et al. Particle size distribution and optimal capture of aqueous microbial eDNA. Methods Ecol Evol. 2014;5:676–684. https://doi.org/10.1111.2041-210X.12206
- Majaneva M, Diserud OH, Eagle SHC, Bostrӧm E, Hajibabaei M, et al. Environmental DNA filtration techniques affect recovered biodiversity. Sci Rep. 2018;8:4682. https://doi.org/10.1038/s41598-018-23052-8