Filter pore size
Commonly used filter pore sizes for eDNA studies range from 0.22 µm and 0.45 µm on the smaller side up to 10 µm on the large side. The smaller sizes are more traditional in the sense that, historically, aquatic DNA samples were collected to interrogate the microbial communities in samples, since they are not something that can be easily differentiated by eye or microscopy and 0.22 µm does a good job of physically capturing aquatic microbes. However, given that most of the biological material in aquatic samples is microbial in origin, this also biases the DNA captured to be more microbial in origin. If a larger pore size is chosen, for example 1 µm, most of the bacteria will pass through the filter and predominantly eukaryotic DNA will be captured. However, protists (single-celled eukaryotes, like phytoplankton) will still be captured and may continue to dominate the DNA captured on the filter. Therefore, some research teams are choosing to use larger pore sized filters to reduce the amount of prokaryotic and protistan DNA on filters while retaining genetic material from larger organisms, like skins cells or fish scales or fecal material. This may improve detection of animals but it does so at the expense of capturing the ‘whole picture’ of biodiversity from microbes to mammals. So, it is important to think critically about what your current and potential target taxa of choice are when choosing filter pore size.
Filter type
There are a variety of filter types available for eDNA studies. The choice of filter type will often be dictated by the filtering apparatus that will be used in your survey. The most common are:
Flat filters - This is the most basic type of filter, consisting of a flat circular disc of filter material. The filter disc needs to be inserted into a filter housing during the filtration step. It can then be preserved for down-stream lab Processing. While these are the cheapest and most basic type of filter. A potential down-side is that extra care must be taken when handling these filters because they require direct handling of the filter material. Sterile forceps and gloves should be used to add/remove them from the filter housing to avoid contamination.
Capsule filters - With capsule filters the filtering material is fully enclosed in a plastic shell and preservatives can be added (using a syringe) into the capsule. The advantage is that this method avoids the need to directly touch the filter, which can help to minimize contamination. The downside is that specialized workflows are required in the lab to extract DNA from the filter within the capsule.
Self-preserving filters - These are a unique type of capsule filter that does not require the addition of a chemical preservative before sample storage. This is a common filter type used in commercially available samples such as the Smith-Root system.
Filtration apparatus
Beyond physically pushing a sample through a filter using a syringe, as described earlier, there are a variety of additional ways to filter aquatic eDNA samples, spanning DIY to commercial approaches.
DIY or Low-Cost Vacuum Pump
A simple vacuum pump system can be assembled using parts that can be purchased at your local hardware store and/or your favorite scientific supply company. Here is an example protocol that employs a garden sprayer to draw a vacuum and pull an aquatic eDNA sample through a Sterivex or other capsule filter. You could alternatively use a hand-operated vacuum pump paired with a vacuum flask and filter funnel fitted with a flat filter as described in this protocol.
DIY or Low-Cost Peristaltic Pump
A lower-cost peristaltic pump system can be built by operating a commercially-available peristaltic pump head using your everyday power drill. Alternatively, you can procure the parts and build a small peristaltic pump system for less than $350 USD. Peristaltic pumps push water through a filter, rather than pull as with vacuum pumps, and are typically a more gentle form of filtration. This is advantageous because it reduces the risk of bursting cells and losing genetic material through the filter.
Commercial Vacuum Pump
Vacuum pumps and filter manifolds can be purchased from commercial suppliers and employed to filter eDNA samples. If paired with a multi-position manifold, multiple eDNA samples can be filtered at once. Here is an example of a vacuum-based eDNA sample filtration protocol.
Commercial Peristaltic Pump
Peristaltic pumps, with compatible pump heads, can be purchased from commercial suppliers. Depending on the type of pump, anywhere from one to four pump heads can be operated simultaneously by the same peristaltic pump drive, allowing multiple samples to be filtered at once. Here is a nice example of a protocol that employs peristaltic pumps.
Gravity Filtration
If you simply won’t have time to filter your samples actively, gravity filtration is another option that is increasingly used, whereby your sample is placed into a sterile IV bag, a filter is attached to the tubing exiting the bag, and then the apparatus is hung up to allow gravity to drive the flow of water through the filter. This approach does take longer compared to filtration using a more mechanical solution but is advantageous for its hands-off nature. Here is an example protocol for this method.
Filter material and Surface area
Additional decisions can be made about the material and actual size (surface area) of the filter. Generally these decisions will be dictated by the apparatus used for filtering or the amount of water that you need to filter (more water can be passed through a filter with a larger surface area).