OTU tablesSequence reference databaseFiles containing the sequences for the reference databases for Batrachia and Teleostei speciesRun1Compressed file containing: 1) Forward sequence file 2) Reverse sequence file 3) File with experiment name, sample name and the tags, the forward primer and the reverse primer sequence associated.Run2Compressed file containing: 1) Forward sequence file 2) Reverse sequence file 3) File with experiment name, sample name and the tags, the forward primer and the reverse primer sequence associated.Run3Compressed file containing: 1) Forward sequence file 2) Reverse sequence file 3) File with experiment name, sample name and the tags, the forward primer and the reverse primer sequence associated.Run4Compressed file containing: 1) Forward sequence file 2) Reverse sequence file 3) File with experiment name, sample name and the tags, the forward primer and the reverse primer sequence associated.Run5Compressed file containing: 1) Forward sequence file 2) Reverse sequence file 3) File with experiment name, sample name and the tags, the forward primer and the reverse primer sequence associated.Run6Compressed file containing: 1) Forward sequence file 2) Reverse sequence file 3) File with experiment name, sample name and the tags, the forward primer and the reverse primer sequence associated.Run7Compressed file containing: 1) Forward sequence file 2) Reverse sequence file 3) File with experiment name, sample name and the tags, the forward primer and the reverse primer sequence associated.Run8Compressed file containing: 1) Forward sequence file 2) Reverse sequence file 3) File with experiment name, sample name and the tags, the forward primer and the reverse primer sequence associated.

Global biodiversity in freshwater and the oceans is declining at high rates. Reliable tools for assessing and monitoring aquatic biodiversity, especially for rare and secretive species, are important for efficient and timely management. Recent advances in DNA sequencing have provided a new tool for species detection from DNA present into the environment. In this study, we tested if an environmental DNA (eDNA) metabarcoding approach, using water samples, can be used for addressing significant questions in ecology and conservation. Two key aquatic vertebrate groups were targeted: amphibians and bony fish. The reliability of this method was cautiously validated in silico, in vitro, and in situ. When compared with traditional surveys or historical data, eDNA metabarcoding showed a much better detection probability overall. For amphibians, the detection probability with eDNA metabarcoding was 0.97 (CI = 0.90-0.99) versus 0.58 (CI = 0.50-0.63) for traditional surveys. For fish, in 89% of the studied sites, the number of taxa detected using the eDNA metabarcoding approach was higher or identical to the number detected using traditional methods. We argue that the proposed DNA-based approach has the potential to become the next-generation tool for ecological studies and standardized biodiversity monitoring in a wide range of aquatic ecosystems.