A validation scale to determine the readiness of environmental DNA assays for routine species monitoring

Abstract

1. Environmental DNA (eDNA) analysis utilises trace DNA released by organisms into their environment for species detection and is revolutionising non-invasive species monitoring. The use of this technology requires rigorous validation - from field sampling to interpretation of PCR-based results - for meaningful application and interpretation. Assays targeting eDNA released by individual species are typically validated with no predefined criteria to answer specific research questions in one ecosystem. Their general applicability, uncertainties and limitations often remain undetermined. The absence of clear guidelines prevents targeted eDNA assays from being incorporated into species monitoring and policy, thus their establishment will be key for the future implementation of eDNA-based surveys.

2. We describe the measures and tests necessary for successful validation of targeted eDNA assays and the associated pitfalls to form the basis of guidelines. A list of 122 variables was compiled and consolidated into a scale to assess the validation status of individual assays. These variables were evaluated for 546 published single-species assays. The resulting dataset was used to provide an overview of current validation practices and test the applicability of the validation scale for future assay rating.

3. The 122 variables representing assay validation status were classified into 14 thematic blocks, such as “in silico analysis”, and arranged on a 5-level validation scale from “incomplete” to “operational”. Additionally, minimum validation criteria were defined for each level. The majority (30%) of investigated assays were classified as Level 1 (incomplete), and 15% did not achieve this first level. These assays were characterised by minimal in silico and in vitro testing, but their share in annually published eDNA assays has declined since 2014. The total number of reported variables ranged from 20% to 76% and deviated both between and within levels.

4. The meta-analysis demonstrates the suitability of the 5-level validation scale for assessing targeted eDNA assays. It is a user-friendly tool to evaluate previously published assays for future research and routine monitoring, while also enabling appropriate interpretation of results. Finally, it provides guidance on validation and reporting standards for newly developed assays.