One size does not fit all: Tuning eDNA protocols for high‐ and low‐turbidity water sampling

Abstract

Findings from eDNA metabarcoding are strongly influenced by experimental approach, yet the effect of pre-PCR sample processing on taxon detection and estimates of biodiversity across different water types is still poorly resolved. To fill this data gap, we investigated the impact of sampling effort, extraction method, and filter pore size on DNA yield, PCR inhibition, and 16S rDNA metabarcoding results for fishes in water samples collected from inshore turbid-and offshore clear-water environments. The turbid-water samples had high concentrations of suspended organic and/ or inorganic material and yielded ~3.2× more DNA and exhibited high levels of PCR inhibition compared with the low-turbidity, clear-water samples. Importantly, there were no striking differences in the results of our metabarcoding experiments based on extraction method or filter pore size. While a small number of unique species of relatively low read count were detected in all turbid-water treatments, most species were consistently detected across samples. Results for the clear-water samples were strikingly different, with low DNA yield, high levels of variation across replicates, and a high number of non-overlapping species across treatments. These findings indicate a patchy distribution of eDNA in offshore environments, which means higher volumes of water (≥ 2 L per replicate) must be filtered in habitats where target DNA is likely to be sparse. In semi-closed systems such as estuaries, higher concentrations of target DNA are expected, and we found that either a 1.0 or 3.0 µm filter pore size was sufficient to capture standing diversity, while decreasing the risk of clogging. For economical DNA extraction and inhibitor removal, we recommend a combination of Omega Bio-tek E.Z.N.A Tissue DNA kit followed by a PCR inhibitor removal step using the Zymo Kit. Finally, we emphasize that pilot studies should be undertaken whenever sampling in a new environment to identify which protocol is most appropriate.

Fine-tuning the performance of abundance estimation based on environmental DNA (eDNA) focusing on eDNA particle size and marker length

“…Alternatively, different physiological origins and physiochemical structures of eDNA might result in its different persistence and spatial dispersion, hence influencing the relationship between eDNA concentration and species abundance. Additionally, the >3 μm pore size filter generally reduces filter clogging, increasing the filtration volume of the water samples considerably compared with smaller pore size filters (e.g., Kumar et al, 2021). Moreover, Takasaki et al (2021) recently compared the pre-filtration performance on eDNA detection using various pore size filters (10-840 μm pore sizes).…”

Optimizing an enclosed bead beating extraction method for microbial and fish environmental DNA

“…Ultimately, it may be important to adjust sampling strategies to accurately monitor fish and other vertebrate DNA in dynamic coastal habitats (Bessey et al, 2020; Miya et al, 2020). For instance, recent studies have suggested that increasing filtration volumes to >2 L per filter may be necessary to capture fish biodiversity (Bessey et al, 2020; Kumar et al, 2021), although such work has not been systematically tested for Sterivex filters and requires optimization based on sampling environment to avoid filter clogging or PCR inhibition (Kumar et al, 2021). In addition to increased filtration volumes, fish biodiversity monitoring may be improved with higher spatial resolution or by increasing the number of technical replicates (e.g., 4–6 filters).…”

“…Even so, eDNA sampling has become an important technique to monitor invasive (or non-native) aquatic organisms (Robson et al, 2016;Ruppert et al, 2019). Although efforts to characterize marine life via eDNA metabarcoding have recently been employed off coastal Florida (Ames et al, 2021;Kumar et al, 2021;Sawaya et al, 2019), 12S MiFish primers have not yet been used to identify fish in this region. To establish a baseline understanding of fish observed in our eDNA dataset, including several taxa that stood out as non-native (sal- (Berkeley & Cantillo, 2004;Roessler, 1965;Serafy et al, 2003).…”

“…Optimal filtration and extraction methods also depend on the sampling environment. For instance, coastal areas with high microbial biomass and particulate matter may limit filtration volumes and increase the risk of PCR inhibition, while offshore areas may have more dilute DNA concentrations and require greater filtration volumes (Kumar et al, 2020(Kumar et al, , 2021Li et al, 2018;Tsuji et al, 2019). Sterivex filters have become an increasingly popular method to collect marine eDNA, as they are simple to use, can be employed in the field on research cruises, and can be immediately fixed (or frozen) on site to avoid DNA degradation (Bryant et al, 2016;Ushio, 2019;Wong et al, 2020).…”

Metabarcoding the marine environment: from single species to biogeographic patterns

“…Differences in sampling and laboratory protocols, as well as methods of data analyses, challenge experimental replication and cross-study interpretation. In this special issue, Kumar et al (2021) contribute one of only a few published studies that employ replicated experiments designed specifically to assess the impact of sampling effort, DNA extraction method, and filter pore size on DNA yield, PCR inhibition, and metabarcoding results (16S rDNA for fishes).…”