Recommendations for analytical procedures and future experiments
A growing body of literature demonstrates that eDNA-based detection is a powerful, sensitive and non-invasive method of biodiversity detection, yet the extent to which existing methods may be susceptible to inefficiencies remains to be systematically investigated. It is evident that at least four states of eDNA exist and not all of them may be captured by the various combinations of methods (Figure 3) used to isolate DNA from water.
To maximize detection rates, methods that capture and isolate DNA from all states should ideally be utilized, such as water filtration using different pore sizes (i.e., to capture particle bound or cellular DNA and avoid clogging the filter) followed by precipitation of the filtrate (i.e., to capture dissolved DNA). Specifically, adsorption effects should be considered when capturing and extracting eDNA from turbid waters, or in the presence of highly concentrated suspended solids as on a filter. A side effect of DNA extraction is the release of intracellular DNA during cell lysis which could encounter positively charged mineral surfaces that were co-captured during filtration, resulting in the newly released DNA becoming particle bound during extraction and subsequently reduced DNA yield. In such cases, extraction buffers that effectively extract DNA from mineral surfaces will need to have the corresponding compositions to favour desorption and prevent adsorption of DNA liberated from cells. In particular, these extraction buffers should: (i) have a sufficiently high (i.e., alkaline, pH 9-10) pH to result in DNA-sorbent electrostatic repulsion, but not too high to facilitate base-catalysed DNA backbone hydrolysis, (ii) contain competing co-adsorbates such as phosphate, pentaphosphate, or possibly a DNA molecule that does not contain the targeted sequence of the analyte DNA, and (iii) contain complexing agents for divalent cations to minimize the possibility of cation bridging of DNA to negatively charged sorbent surfaces. Notably, extraction protocols developed for soil and sediment may be more efficient for the extraction of eDNA from water with a high concentration of particles whose surfaces can adsorb DNA, particularly if these particles are concentrated with eDNA during filtration64. A systematic DNA extraction assessment using artificial control samples with known concentrations of freely dissolved, particle-adsorbed and intracellular DNA is needed to determine which states are most efficiently captured by common extraction protocols. This would aid optimization of the extraction protocol and account for the different eDNA states while maximizing their extraction efficiency3,65.
Where possible, we recommend that eDNA practitioners employ methods to capture multiple states of eDNA. All samples should then be combined for analysis or analysed independently if eDNA states are likely to influence the research or management questions under investigation, (e.g., inferences of where and when a species was present3). If it is impossible to extract all eDNA states at every study site, we recommend that eDNA practitioners resample sites that are suspected false negatives should their chosen methods of eDNA capture and extraction (most likely filtration and a commercial DNA extraction kit) fail to produce eDNA detections. However, a caveat to the above is that if different states have different decay rates (e.g., particle bound DNA might persist longer than dissolved DNA), then the time and space inference as to when a species was present in the sampled environment becomes less clear. Thus, for accurate inferences of time and space, not just detection, more research is required to determine concentration dynamics for all eDNA states present in different ecosystems3.