Evaluation of detection probabilities at the water-filtering and initial PCR steps in environmental DNA metabarcoding using a multispecies site occupancy model.

Environmental DNA (eDNA) metabarcoding is a recently developed method to assess biodiversity based on a high-throughput parallel DNA sequencing applied to DNA present in the ecosystem. Although eDNA metabarcoding enables a rapid assessment of biodiversity, it is prone to species detection errors that may occur at sequential steps in field sampling, laboratory experiments, and bioinformatics. In this study, we illustrate how the error rates in the eDNA metabarcoding-based species detection can be accounted for by applying the multispecies occupancy modelling framework. We report a case study with the eDNA sample from an aquarium tank in which the detection probabilities of species in the two major steps of eDNA metabarcoding, filtration and PCR, across a range of PCR annealing temperatures, were examined. We also show that the results can be used to examine the efficiency of species detection under a given experimental design and setting, in terms of the efficiency of species detection, highlighting the usefulness of the multispecies site occupancy modelling framework to study the optimum conditions for molecular experiments.