How do we create analytical controls for state?
The importance of appropriate analytical controls in eDNA research is well established (e.g.,13,61). These include field and laboratory controls that are designed to assess contamination (negative controls)62, analytical precision (biological and technical replicates), and sensitivity (positive controls). However, these controls do not account for eDNA being present in different states nor do these controls allow assessment of whether eDNA in each state(s) is accurately quantified. Moreover, incomplete recovery of analytical controls typically leads to the conclusion that PCR inhibition is involved. While this clearly is a possibility, we propose that results could also be confounded because current protocols may not completely extract DNA from all four states if present in the sample. Therefore, additional analytical controls are needed to disambiguate the cause of observed signal attenuation (e.g., PCR inhibition versus inefficient extraction across states).
There are various analytical controls employed in the eDNA literature, but these are inconsistently applied. Some researchers (e.g.,63) advocate multiplexing an assay for a given target species together with an assay designed to detect a co-occurring species presumed to be ubiquitous in the environment, such as algae (e.g., using a generalized plant chloroplast DNA assay), to demonstrate that the PCR reaction was not inhibited. Yet, because the state (Figure 1) and concentration of any species’ eDNA is unknown, it cannot be used to assess relative rates of PCR inhibition and/or inefficient eDNA recovery. To address this issue, internal standards of known DNA concentration and state could be applied at various stages in the workflow (Figure 4). Synthetic DNA has been used as an internal positive control to quantitate the relative degree of PCR inhibition, but this does not account for inefficient extraction of different eDNA states. Applying a “spike in” control prior to the extraction/precipitation step could result in some sorption of the control DNA, but again the attenuation of the PCR signal could not be used to discriminate between inhibition and inefficient recovery.
Developing analytical controls to assess whether eDNA is bound to cellular debris, adsorbed to particles or dissolved in solution remains a challenge. Size fractionation can be achieved by filtering a sample through multiple filters of progressively smaller pore size and subsequently extracting eDNA from each individual filter and the filtrate. Assuming any DNA that passed through the filters into the filtrate represents dissolved eDNA and potentially even particles, it is possible to quantify this pool. However, eDNA recovered from the filters cannot be separated into cellular bound vs. particle bound DNA without utilizing extraction protocols optimized to recover only particle bound or cellular debris bound eDNA. Protocols for separating soluble DNA (i.e., extracellular and bound to particles) from insoluble DNA (i.e., still inside the cell) have been developed60. Their parallel application to known mixtures of cellular bound, particle bound and dissolved eDNA could prove illuminating by separating out the different states and analysing them separately for detection of a target species or community. However, quantifying the eDNA in each category before assembling the mixtures would be non-trivial and even then, the approach could not easily assess the dynamic conversion of DNA between states that may occur during the extraction. These issues notwithstanding, the combined use of cellular material, plasmids (e.g. as surrogates for organelles), synthetic DNA and varied adsorbent materials, together with size fractionation and multiple extraction techniques as applied across a gradient of environmental conditions could yield novel insights concerning extraction efficiency among eDNA states and the dynamic conversion processes between them when selectively applied to each sample processing step (Figure 4).