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.