Figure 3: Sankey diagram summarising water preservation, capture, filter preservation, extraction (lysis and inhibitor removal), and detection methods used by studies included in our literature review.
Most assays (n = 100) targeted fish (49.0%), followed by crustaceans (17.0%) (Figure S3a). The majority of assays (n = 123) were used for quantification (39.8%) or detection (35.8%) (Figure S3b), and used in the Palearctic (45.3%) or Nearctic (40.7%) (Figure S3d). Assays were usually deployed in natural environments (n = 84), specifically lentic (20.7%) or lotic (26.5%) freshwater systems (Figure. S3c). Where assays were used in experimental systems (classed as ‘other’ for environment), the volume of these artificial water systems ranged from 900 ml to 336,000 L (median = 20 L). Sample sizes (i.e., number of sampling sites) ranged from 1 to 197 (median = 4), with between 1 and 120 biological replicates (median = 5) taken, and between 15 ml and 6 L of water (median = 500 ml) collected per biological replicate.
The broad workflows used by assays included in our literature review are summarised in Figure 3. Most assays used cooling (n = 25) after ‘other’ (n = 58, typically centrifugation or resin beads) for water sample preservation, followed by filtration (n = 126; Figures 3, S4) for eDNA capture. Ethanol/sodium acetate (n = 5) for water sample preservation followed by precipitation (n = 31) for eDNA capture was less popular but constituted a second major analytical workflow (Figures 3, S4). Of those assays using filtration, glass fibre filter membranes (0.7 μm pore size) were most commonly used, followed by polycarbonate track-etched, cellulose nitrate, nylon and ‘other’ membrane types, including cellulose acetate and polyethersulfone (Figure S5). Filters were typically frozen at -20℃ for preservation of DNA in the retentate (Figure 3), but storage times were often not reported. A full breakdown of precipitation and filtration methods can be found in Supporting Information (Figures S6, S7).
The vast majority of assays (n = 125) used commercial extraction kits (82.0%) as opposed to unbranded protocols (18.0%) (Figure S8), with the Qiagen DNeasy Blood and Tissue Kit being the most commonly used (47.76%; n = 110) (Figure S9). Mechanical disruption with chemicals and chemicals only were secondary to an enzyme with chemicals for cell lysis (Figure 3). Commercial kits typically employed an enzyme with temperature to induce cell lysis and lacked an inhibitor removal step, yet post-extraction inhibitor removal was uncommon (Figure S10). Where post-extraction inhibitor removal was performed, this was either done by phase separation or chemical flocculation (Figure 3) using methods such as the Zymo One Step PCR Inhibitor Removal Kit, the Promega Wizard Genomic DNA Purification Kit, chloroform or dilution.
Most assays (n = 136) targeted mitochondrial genes using quantitative PCR (69.9%) (Figures 3, S11). Typically, three technical replicates were performed in 20 μl reactions using 2 μl of template DNA. The majority of assays (80.0%) did not use an internal positive control to test for inhibition and did not determine the Limit of Detection (54.4%), Limit of Quantification (71.0%) or effects of the environmental matrix (78.0%) (Figure S12). Most assays used commercial master mixes (Figure S13), such as Applied Biosystems TaqMan Environmental Master Mix 2.0 and TaqMan Gene Expression Master Mix (Figure S14), as opposed to custom master mixes. Where custom master mixes were used, MgCl2 concentration ranged from 1.5 to 2.5 (median = 2) and dNTP concentration ranged from 0.05 to 0.25 (median = 0.20). Promega 5x Colorless GoTaq Flexi Reaction Buffer and Promega GoTaq Flexi DNA Polymerase were the most commonly used buffer type and enzyme type respectively. Enhancers were not often added to PCR reactions, but Bovine Serum Albumin (BSA) was most common where enhancers were used (21.3%) (Figure S15).
Crucially, most assays (n = 145) did not measure, record or report environmental parameters that are expected to affect the distribution of DNA among states and determine stability of DNA (Figure S16). Parameters that were recorded and reported included temperature (50.3%), pH (22.0%), UV exposure (9.0%), season (68.0%), canopy cover (3.0%), conductivity/salinity (22.0%), geology of catchment (12.0%) and dissolved oxygen (15.0%). One third of papers would require the authors to be contacted to clarify their analytical workflow or ascertain if they collected environmental data but did not report it (Figure S17).
Taken all together, our meta-analysis suggests that most single-species studies employ methods that analyse a similar and potentially restricted state of eDNA. The majority of studies use filtration at pore sizes through which most dissolved eDNA may pass if clogging does not occur. After filtration, the eDNA on the filter is isolated with similar lysis methods and purification buffers provided with commercial extraction kits that fundamentally employ similar chemistry (see Table S2). Most of these commercial kits likely do not promote particle bound DNA to desorb. To be certain, the constituents of the buffers would need to be determined which was not feasible since most of these are trade secrets. If these commercial kit buffers do not have competitive binders and do not reach a pH high enough to promote desorption, it is likely that DNA adsorbed onto particles was not isolated. If these assumptions are true (i.e. dissolved DNA flows through filters and extraction kit buffers do not promote desportion), then eDNA detections from the studies reviewed here may originate from only inter-cellular or organellar DNA. However, extensive research comparing whether specific molecular methods co-purify eDNA states would be needed to verify this claim.