Time to get real with qPCR controls: The frequency of sample
contamination and the informative power of negative controls in
environmental (e)DNA studies
Abstract
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 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
lab experiments to (1) 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, (2) determine the rate at which negative controls
(i.e., “blanks”) detect varying concentrations of systemic
contamination, (3) 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.