Capturing the eDNA they left behind

Without a sample filtration/concentration step, would eDNA be lost in the environment?

This is a plausible question, and in fact most eDNA protocols use a filtration or precipitation step to concentrate the sample in the sample collection stage. In part 1 of this 3-part eDNA filtration blog series, we discuss why sample filtration rather than precipitation is the preferred method in the sample prep stage prior to eDNA extraction.

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 but Hinloet al., 2017 investigated filtering samples through a 47mm, 0.8µm cellulose nitrate filter paper using Pall’s filter funnel manifold and a pump in a set-up similar to figure 1 and compared it to a precipitation method and found that the method involving filtration yielded the highest quantities of DNA⁽¹⁾.

Figure 1: Frequently used filtration workflow set-ups in eDNA sampling.

Time of Filtration and Storage

Environmental DNA degrades readily in the environment, and the rate of eDNA degradation increases with higher temperatures and exposure to UV light. Therefore, it is important to reduce the time between sampling and filtering to retrieve as much eDNA as possible from the sample.

For on-site sampling, many researchers 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  [Figure 1]. Pall’s Sentino® pump and manifold were used in the Nature Research report by Majanevaet al., 2018⁽²⁾, 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 minimizes time for eDNA decay in the samples⁽²⁾. Pall’s Sentino pump is ideal 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.

To learn more about how additional Pall’s product have been integrated into eDNA workflows by the scientific community, please read the full scientific brief. 

Stay tuned for part 2 of this blog series where we will look at which filtration material and pore size the eDNA community are using.

References

1. Hinlo R, Gleeson D, Lintermans M, Furlan E. (2017) Methods to maximise recovery of environmental DNA from water samples. PLoS ONE 12(6): e0179251. 
2. Majaneva, M., Diserud, O.H., Eagle, S.H.C. et al. (2018) Environmental DNA filtration techniques affect recovered biodiversity. Sci Rep 8, 4682