Direct comparison of eDNA capture and extraction methods through measuring recovery of synthetic DNA cloned into living cells

Abstract

Before eDNA surveys can be deployed, the methods used to capture and extract DNA need to be determined. Multiple widely used eDNA capture and DNA extraction methods are available, and the effectiveness of these methods has been assessed in the literature. These studies used raw estimates of target cells captured from mesocosms and tanks, or already extracted DNA, which are not reflective of environmental DNA or precise enough to accurately compare eDNA capture and DNA extraction methods. Here, using qPCR quantification, we compared two eDNA capture and extraction methods using low and high concentrations of DNA cloned into living cells. By cloning target DNA into living cells, we can control the quantity of our starting copy number to directly compare the efficiency and effectiveness of each method throughout the process of eDNA handling and data analysis. This approach is useful for determining which capture and extraction method is suitable for the target species and sampling location of interest before full‐scale deployment. Although our target species DNA is consistently and accurately detected (even at low concentrations), stochasticity is a significant factor in our ability to consistently recover all the starting material by the end of sample processing. By recognizing that stochasticity is important for eDNA recovery, more accurate sampling models and eDNA processing protocols can be developed to increase eDNA detection sensitivity.

Efficacy of Florfenicol and Oxytetracycline Administered in Feed to Control Cisco Mortality Associated with Aeromonas salmonicida Infections

“…The aliquot chosen for infection was identified as 10 7 CFU/mL based on quantification cycle (C q ) values that were quantified using an intraplate, five-point standard curve consisting of 6,250, 1,250, 250, 50, and 10 copies of synthetic gBlock amplicon fragments (Integrated DNA Technologies, San Diego, California). Bacterial culture, concentration, and qPCR detection methods were previously optimized through experiments cited by Bockrath et al (2022). The stock tube of 10 7 CFU/mL was used to directly inoculate the infection baths with a final concentration of 10 3 CFU/mL.…”

“…Bacterial culture, concentration, and qPCR detection methods were previously optimized through experiments cited by Bockrath et al. (2022). The stock tube of 10 7 CFU/mL was used to directly inoculate the infection baths with a final concentration of 10 3 CFU/mL.…”

Validation of a nutria (Myocastor coypus) environmental DNA assay highlights considerations for sampling methodology

“…Water samples are often not only utilized for monitoring in aquatic systems (e.g., brook trout [ Salvelinus fontinalis ]; Wilcox et al, 2016) but have also been successfully applied to terrestrial mammals (e.g., invasive wild pig [ Sus scrofa ]; Williams et al, 2018) and semiaquatic species (e.g., Burmese python [ Python bivittatus ], river otter [ Lontra canadensis ], and Japanese water shrew [ Chimarrogale platycephalus ]; Padgett‐Stewart et al, 2016; Piaggio et al, 2014; Yonezawa et al, 2020). In freshwater habitats, filtration via handheld or backpack pumps is the most common approach for eDNA sampling (e.g., Carim, McKelvey, et al, 2016) but direct sampling (i.e., scooping water into a plastic bottle) followed by centrifugation, chemical precipitation, or mechanical concentration is also common (Bockrath et al, 2022; Piaggio, 2021).…”

Temperature moderates eDNA–biomass relationships in northern pike

“…Although linear relationships have been obtained in controlled experiments, the precision of these estimates varies greatly in natural systems where eDNA on average explains 57% of the variance compared with 81% in controlled mesocosm experiments (Yates et al, 2019). The variability in these estimates can be attributed to differences in ground truthing methods (Rourke, Fowler, et al, 2022), hydrologic conditions (Song et al, 2017), DNA extraction methods (Bockrath et al, 2022; Karlsson et al, 2022), presence of polymerase chain reaction (PCR) inhibiting humic and tannic acids (Lance & Guan, 2020), sediment particles in the water (Stoeckle et al, 2017), size distribution of the local population (Yates, Cristescu, & Derry, 2021; Yates, Wilcox, et al, 2021) and ambient temperature (Lacoursière‐Roussel et al, 2016). Here, two of these factors are focused on, namely, temperature (DNA shedding and degradation) and hydrology (distribution and dilution).…”