Abstract
Environmental DNA (eDNA) analysis has gained traction as a precise and cost-effective method for species and waterways management. To date, publications on eDNA protocol optimization have focused primarily on DNA yield. Therefore, it has not been possible to evaluate the cost and speed of specific components of the eDNA protocol, such as water filtration and DNA extraction method when designing or choosing an eDNA protocol. At the same time, these two parameters are essential for the experimental design of a project. Here we evaluate and rank 27 different eDNA protocols in the context of Chinook salmon (Oncorhynchus tshawytscha) eDNA detection in an estuarine environment. We present a comprehensive evaluation of multiple eDNA protocol parameters, balancing time, cost and DNA yield. We collected samples composed of 500 mL estuarine water from Deverton Slough (38°11'16.7"N 121°58'34.5"W) and 500 mL from tank water containing 1.3 juvenile Chinook Salmon per liter. Then, we compared extraction methods, filter types, use of inhibitor removal kit for DNA yield, processing time, and protocol cost. Lastly, we used an MCMC algorithm together with machine learning to understand the DNA yield of each step of the protocol as well as the interactions between those steps. Glass fiber filtration was to be the most resilient to high turbidites, filtering the samples in 2.32 ± 0.08 min instead of 14.16 ± 1.86 min and 6.72 ± 1.99 min for nitrocellulose and paper filter N1, respectively. The filtration DNA yield percentages for paper filter N1, glass fiber, and nitrocellulose were 0.00045 ± 0.00013, 0.00107 ± 0.00013, 0.00172 ± 0.00013. The DNA extraction yield percentage for QIagen, dipstick, NaOH, magnetic beads, and direct dipstick ranged from 0.047 ± 0.0388 to 0.475 ± 0.0357. For estuarine waters, which are challenging for eDNA studies due to high turbidity, variable salinity, and the presence of PCR inhibitors, we found that a protocol combining glass filters, magnetic beads, and an extra step for PCR inhibitor removal, is the method that best balances time, cost, and yield. In addition, we provide a generalized decision tree for determining the optimal eDNA protocol for other studies in aquatic systems. Our findings should be applicable to most aquatic environments and provide a clear guide for determining which eDNA protocol should be used under different study constraints.
Highlights
Environmental management relies heavily on knowledge of the spatial distribution of species
Environmental DNA monitoring has gained traction as one of the most sensitive and cost-effective monitoring methods [1], allowing researchers to better estimate species occupancy rates in a given habitat. environmental DNA (eDNA) refers to genetic material present in an environmental sample, which is shed from all organisms in the form of fluids, skin cells/scales, decay and feces from the target species
Filtration or precipitation procedures can isolate eDNA or cells containing DNA from an environmental sample [2]. eDNA acts as a speciesspecific footprint, which can be detected by researchers and used to infer the presence of a target species [3]. eDNA might be used to monitor biodiversity of whole communities as all living beings possess and shed DNA
Summary
Environmental management relies heavily on knowledge of the spatial distribution of species. Environmental DNA (eDNA) monitoring has gained traction as one of the most sensitive and cost-effective monitoring methods [1], allowing researchers to better estimate species occupancy rates in a given habitat. EDNA refers to genetic material present in an environmental sample, which is shed from all organisms in the form of fluids, skin cells/scales, decay and feces from the target species. Filtration or precipitation procedures can isolate eDNA or cells containing DNA from an environmental sample [2]. DNA is present in most environmental samples, chemical factors and physical forces can reduce with the DNA detection of an eDNA assay. Due to high variability in the physical and chemical characteristics of studied environments, there are no clear guidelines to assist investigators in choosing an optimal protocol for their particular eDNA monitoring studies. While we focus on the estuarine habitat here, we assume that if our DNA amplification-based experiments work in these complex conditions, the same approach could be applied to less turbid freshwater and marine conditions
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