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

Environmental DNA metabarcoding represents a powerful and cost-efficient approach for benthic microbial surveys, but is limited by the lack of absolute abundance information, and by the conflation of contemporary and legacy DNA. In this study, we explored the combined use of a propidium-monoazide (PMA) treatment, 16S rRNA ddPCR and metabarcoding to assess the microbial density, composition and diversity of contemporary communities, and estimate the proportion of legacy DNA in deep-sea surface sediment. We used spike-in synthetic DNA to assess the efficiency of the PMA treatment and validated results against flow cytometry (FC) and environmental data. Overall, the efficiency of the PMA treatment was moderate (mean of 45%) and highly correlated with the proportion of extracellular DNA in the samples (r = -0.95). Microbial density estimates from ddPCR correlated weakly with FC results (r = 0.25), but strongly with sediment depth (r = -0.5) and dissolved oxygen (r = 0.56). Accounting for PMA treatment efficiency, legacy DNA amounted to approximately half of total microbial DNA. Comparison of eDNA, eRNA and PMA-treated eDNA (iDNA) microbial assemblages indicated substantially stronger correlation of the later with environmental data, followed by eDNA. Overall, this study provides encouraging results for the application of PMA treatment on sediment samples and the use of ddPCR-based microbial density estimates, but also highlight the need for protocol optimization and further validation to obtain better performance and further confidence.