A field-deployable eDNA metabarcoding workflow including de novo reference assembly for characterizing 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 enable 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 practicality and versatility of this novel workflow. De novo generation of a reference sequence library significantly improved the accuracy and taxonomic resolution of sequence assignments from eDNA samples, particularly on the species level, in turn allowing a characterization 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 bridge data gaps in molecular biodiversity monitoring, providing a scalable solution for real-time 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.