Development and validation of a ddPCR & viability PCR assay for enhancing eDNA benthic surveys: A deep-sea case study

Area of Science:

• Marine microbiology
• Molecular ecology
• Environmental genomics

Background:

• Environmental DNA (eDNA) metabarcoding is cost-efficient for benthic microbial surveys but struggles with absolute abundance and distinguishing contemporary from legacy DNA.
• Propidium monoazide (PMA) treatment, 16S rRNA digital droplet PCR (ddPCR), and metabarcoding offer potential solutions to these limitations.

Purpose of the Study:

• To assess microbial density, composition, and diversity of contemporary communities in deep-sea sediment.
• To estimate the proportion of legacy DNA using combined PMA treatment, ddPCR, and metabarcoding.
• To validate the approach against flow cytometry (FC) and environmental data.

Main Methods:

• Application of PMA treatment to differentiate viable from non-viable microbial cells.
• Quantification of microbial DNA using 16S rRNA ddPCR.
• Metabarcoding analysis of microbial community composition.
• Validation using spike-in synthetic DNA, FC, and correlation with sediment depth and dissolved oxygen.

Main Results:

• PMA treatment efficiency was moderate (45%) and correlated with extracellular DNA levels.
• Microbial density estimates from ddPCR showed weak correlation with FC but strong correlation with sediment depth and dissolved oxygen.
• Legacy DNA constituted approximately half of the total microbial DNA.
• PMA-treated eDNA (iDNA) microbial assemblages showed stronger correlation with environmental data compared to eDNA.

Conclusions:

• The combined use of PMA treatment and ddPCR shows promise for assessing contemporary microbial communities and DNA in marine sediments.
• Further protocol optimization and validation are needed for enhanced accuracy and confidence in deep-sea microbial ecology studies.