Evaluating the effects of laboratory protocols on eDNA detection probability for an endangered freshwater fish

The effectiveness and accuracy of detection using environmental DNA (eDNA) is dependent on understanding the influence laboratory methods such as DNA extraction and PCR strategies have on detection probability. Ideally choice of sampling and extraction method will maximize eDNA yield and detection probability. Determining the survey effort required to reach a satisfactory detection probability (via increased PCR replicates or more sampling) could compensate for a lower eDNA yield if the sampling and extraction method has other advantages for a study, species or system. I analysed the effect of three different sampling and extraction methods on eDNA yield, detection probability and PCR replication for detecting the endangered freshwater fish Macquaria australasica from water samples. The impact of eDNA concentration, PCR strategy, target amplicon size and two marker regions: 12S (a mitochondrial gene) and 18S (a nuclear gene) was also assessed. The choice of sampling and extraction method and PCR strategy, rather than amplicon size and marker region, had the biggest effect on detection probability and PCR replication. The PCR replication effort required to achieve a detection probability of 0.95, ranged from 2 to 6 PCR replicates depending on the laboratory method used. As all methods yielded eDNA from which M. australasica was detected using the three target amplicons, differences in eDNA yield and detection probability between the three methods could be mitigated by determining the appropriate PCR replication effort. Evaluating the effect sampling and extraction methods will have on the detection probability and determining the laboratory protocols and PCR replication required to maximize detection and minimize false positives and negatives is a useful first step for eDNA occupancy studies.