Establishing detection thresholds for environmental DNA using receiver operator characteristic (ROC) curves

Environmental DNA (eDNA) detection is increasingly being used to assess the potential presence of aquatic species. Setting defensible thresholds for qPCR assays are necessary to differentiate “detection” from “non-detection”, but threshold rationales are rarely reported in eDNA studies. Detection thresholds for data inclusion and exclusion have implications for downstream data analysis; uncritical acceptance could result in false positive results (Type I error), whereas overly conservative thresholds could result in high false negative interpretation (Type II error). We assessed the use of the receiver operator characteristics (ROC) framework to identify a detection threshold for balancing sensitivity and specificity of eDNA data. The ROC framework was tested using empirical eDNA data from redside dace (Clinostomus elongatus), an endangered freshwater fish in southwestern Ontario. Based on negative controls, positive controls, and quality assurance guidelines, a minimum concentration of 1 copy reaction−1 was identified as the most appropriate threshold for this study. Although optimal thresholds will likely vary between studies and labs based on empirical data, the ROC framework provides a statistical method for establishing defensible thresholds for data acceptance or rejection as well as tradeoffs between the sensitivity and specificity of eDNA data.

环境DNA（eDNA）检测正日益被用于评估水生物种的潜在存在状况。为实时定量PCR（qPCR）实验设定合理的判定阈值，以区分“检出”与“未检出”结果是必要的，但eDNA相关研究中极少报告阈值的制定依据。用于数据纳入与排除的检出阈值会对后续数据分析产生影响：不加批判地接受阈值可能导致假阳性结果（I型错误），而过于保守的阈值则可能引发高比例的假阴性解读（II型错误）。我们评估了采用受试者工作特征（ROC）框架来确定可平衡eDNA数据灵敏度与特异度的检出阈值的可行性。本研究采用产自安大略省西南部的濒危淡水鱼类红侧雅罗鱼（Clinostomus elongatus）的实测eDNA数据对ROC框架进行了测试。基于阴性对照、阳性对照及质量保证指南，本研究确定1个拷贝·反应⁻¹的最低浓度为最合适的判定阈值。尽管基于实测数据的最优阈值可能因研究与实验室而异，但ROC框架为建立合理的数据纳入/排除阈值，以及权衡eDNA数据的灵敏度与特异度提供了一种统计学方法。