Journal: Ecology and Evolution

Article Title: Addressing a Flat‐Out Problem: Environmental DNA ( eDNA ) Exposes Silent Infestations of Acropora ‐Eating Flatworms ( Prosthiostomum acroporae ) in Coral Aquaculture

doi: 10.1002/ece3.73386

Figure Lengend Snippet: Specificity against non‐target species gDNA via droplet digital PCR assay. Left panels contain species of common occurring aquarium invertebrates, labeled at lowest possible taxonomic resolution. Right panel shows closely related polyclad flatworm species. Specimen information for non‐target species in both panels is listed in Appendix . The number in bracket refers to specimen ID (Table , Appendix ). The pink line indicates the threshold for positive droplets set based on the positive droplet clustering of the positive control P. acroporae (“AEFW”, specimen ID 1, Table ). All samples were tested in ddPCR technical replicates. NTC, no template control.

Article Snippet: As all non‐target matches displayed either at least four mismatches, had low percent identity to the probe sequence, or did not occur within target species range, the assay was considered specific to P. acroporae . ddPCR optimisation resulted in a 25 μL reaction volume containing the following: 12.5 μL Bio‐Rad ddPCR Supermix for Probes (no dUTP), 900 nM each of forward and reverse primers, 100 nM of a hydrolysis TaqMan probe, 5 μL DNA template, and PCR‐grade pure water (Thermo Fisher Scientific) to volume (Appendix ).

Techniques: Digital PCR, Labeling, Positive Control, Control