A Field‐Deployable <scp>eDNA</scp> Metabarcoding Workflow Including De Novo Reference Assembly for Characterising Understudied Biodiversity Hotspots

Field-deployable DNA metabarcoding offers a transformative approach to biodiversity research and monitoring, yet its application remains limited due to technical constraints and a lack of reference data in poorly studied ecosystems. Combining isothermal Recombinase Polymerase Amplification (RPA) and Oxford Nanopore sequencing, we introduce a two-step approach that uses non-invasive species barcoding to directly generate reference sequences for use in environmental DNA (eDNA) metabarcoding, and enables real-time, PCR-free and cost-effective molecular assessment of ecological communities in the field. Using an endemic and understudied tropical amphibian assemblage as a model, we demonstrate the functionality of this novel workflow. De novo generation of a reference sequence library from amphibian skin swab samples significantly improved the accuracy and taxonomic resolution of sequence assignments from eDNA samples, particularly on the species level, in turn allowing a characterisation of fine-scale patterns in community composition. Beyond generating new RPA-compatible amphibian metabarcoding primers, our results show that combining field-based eDNA metabarcoding with the offline assembly of a local reference database can directly bridge existing data gaps in molecular biodiversity monitoring, providing a scalable solution to accelerate biodiversity assessments in data-deficient ecosystems. This workflow paves the way for broader deployment of molecular tools in global biodiversity hotspots-particularly in remote and resource-limited tropical regions-to directly contribute critical baseline data, and support conservation efforts in regions where they are most urgently needed.