Abstract
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.
Highlights
Environmental (e)DNA refers to sampling and detection techniques for deoxyribonucleic acid (DNA) released by organisms into the environment
polymerase chain reaction (PCR) controls were reported in 71%, field controls were reported in 51%, extraction controls were reported in 36%, and DNA capture controls were reported in 25% of the 100 targeted eDNA studies that collected negative controls (Figure 2)
PCR controls were reported in 71%, extraction controls were reported in 44%, and field controls and DNA capture controls were each reported in 20% of the 33 metabarcoding studies that collected negative controls (Figure 2)
Summary
Environmental (e)DNA refers to sampling and detection techniques for deoxyribonucleic acid (DNA) released by organisms into the environment (e.g., water, soil, or air). Because eDNA Contamination most eDNA methods utilize polymerase chain reaction (PCR) of relatively short DNA fragments (generally < 200 nucleotides), these methods are sensitive enough to detect DNA at extremely low concentrations. This sensitivity is a key advantage, as it allows users to make inferences about taxa presence even when they are at abundances too low to be detected by traditional, nonmolecular techniques. This extreme sensitivity presents a challenge, as it heightens susceptibility to contaminating DNA. Effective means for detecting contamination are needed to inform potentially costly management decisions, but to identify and strengthen weak points in current workflow protocols
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