Time to get real with qPCR controls: The frequency of sample contamination and the informative power of negative controls in environmental DNA studies.

Environmental (e)DNA methods have enabled rapid, sensitive and specific inferences of taxa presence throughout diverse fields of ecological study. However, use of eDNA results for decision-making has been impeded by uncertainties associated with false positive tests putatively caused by sporadic or systemic contamination. Sporadic contamination is a process that is inconsistent across samples and systemic contamination occurs consistently over a group of samples. Here, we used empirical data and laboratory experiments to (i) estimate the sporadic contamination rate for each stage of a common, targeted eDNA workflow employing best practice quality control measures under simulated conditions of rare and common target DNA presence, (ii) determine the rate at which negative controls (i.e., "blanks") detect varying concentrations of systemic contamination, and (iii) estimate the effort that would be required to consistently detect sporadic and systemic contamination. Sporadic contamination rates were very low across all eDNA workflow steps, and, therefore, an intractably high number of negative controls (>100) would be required to determine occurrence of sporadic contamination with any certainty. Contrarily, detection of intentionally introduced systemic contamination was more consistent; therefore, very few negative controls (<5) would be needed to consistently alert to systemic contamination. These results have considerable implications to eDNA study design when resources for sample analyses are constrained. Published 2021. This article is a U.S. Government work and is in the public domain in the USA.