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

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. Before eDNA surveys can be deployed, the methods used to capture and extract DNA need to be determined. Using qPCR quantification, we compared two eDNA capture and extraction methods using low and high concentrations of DNA cloned into living cells to 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.