The Elephant in the Lab (and Field): Contamination in Aquatic Environmental DNA Studies

The rapid evolution of environmental (e)DNA methods has resulted in knowledge gaps in smaller, yet critical details like proper use of negative controls to detect contamination. Detecting contamination is vital for confident use of eDNA results in decision-making. We conducted two literature reviews to summarize (a) the types of quality assurance measures taken to detect contamination of eDNA samples from aquatic environments, (b) the occurrence, frequency and attribution (i.e., putative sources) of unexpected amplification in these quality assurance samples, and (c) how results were interpreted when contamination occurred. In the first literature review, we reviewed 156 papers and found that 91% of targeted and 73% of metabarcoding eDNA studies reported inclusion of negative controls within their workflows. However, a large percentage of targeted (49%) and metabarcoding (80%) studies only reported negative controls for laboratory procedures, so results were potentially blind to field contamination. Many of the 156 studies did not provide critical methodological information and amplification results of negative controls. In our second literature review, we reviewed 695 papers and found that 30 targeted and 32 metabarcoding eDNA studies reported amplification of negative controls. This amplification occurred at similar proportions for field and lab workflow steps in targeted and metabarcoding studies. These studies most frequently used amplified negative controls to delimit a detection threshold above which is considered significant or provided rationale for why the unexpected amplifications did not affect results. In summary, we found that there has been minimal convergence over time on negative control implementation, methods, and interpretation, which suggests that increased rigor in these smaller, yet critical details remains an outstanding need. We conclude our review by highlighting several studies that have developed especially effective quality assurance, control and mitigation methods.