A Field-Deployable eDNA Metabarcoding Workflow Including De Novo Reference Assembly for Characterising Understudied...

Area of Science:

• Molecular Ecology
• Biodiversity Monitoring
• Genomics

Background:

• Field-deployable DNA metabarcoding is limited by technical constraints and lack of reference data in understudied ecosystems.
• Environmental DNA (eDNA) metabarcoding requires robust reference libraries for accurate biodiversity assessments.
• Tropical regions, particularly those with limited resources, face significant data gaps in molecular biodiversity monitoring.

Purpose of the Study:

• To develop a field-deployable, PCR-free workflow for generating reference sequences from environmental DNA (eDNA).
• To enhance the accuracy and taxonomic resolution of eDNA metabarcoding in data-deficient ecosystems.
• To enable real-time, cost-effective molecular assessment of ecological communities in remote tropical regions.

Main Methods:

• Combined isothermal Recombinase Polymerase Amplification (RPA) with Oxford Nanopore sequencing for a two-step approach.
• Generated de novo reference sequence libraries from amphibian skin swab samples.
• Applied field-based eDNA metabarcoding coupled with offline assembly of a local reference database.

Main Results:

• The novel workflow significantly improved accuracy and species-level taxonomic resolution in eDNA sample analysis.
• Successfully characterized fine-scale patterns in amphibian community composition.
• Generated new RPA-compatible amphibian metabarcoding primers.

Conclusions:

• This approach bridges data gaps in molecular biodiversity monitoring, offering a scalable solution for data-deficient ecosystems.
• Facilitates broader deployment of molecular tools in biodiversity hotspots, especially in remote tropical regions.
• Provides critical baseline data to support conservation efforts in under-resourced areas.