Optimization of prokaryotic DNA isolation in the precious red coral Corallium rubrum for metagenomic analyses

Summary

The diversity of the bacterial communities associated with Corallium rubrum is increasingly well characterized (Van de Water et al. 2016; 2024; Prioux et al. 2023). Notably, C. rubrum microbiome is dominated by bacteria affiliated with the phylum Spirochaetota and exhibits a remarkable stability across both space and time (van de Water et al., 2016; 2018). Despite these taxonomic insights, the functional roles of these microorganisms within the holobiont remain largely unknown. To address this gap, we seek to 1) reconstruct metagenome-assembled genomes (MAGs) of the dominant taxa and 2) investigate their functional roles in C. rubrum.

A key difficulty in conducting metagenomic analyses on holobionts, compared to free-living microbes, is the predominance of host-derived DNA in the samples. After sequencing total genomic DNA, most of the reads are usually derived from the host genome (often more than 98%), with only a small proportion corresponding to microbial sequences. Moreover, the selective isolation of microbial DNA from a mixed pool of host and microbial DNA is technically demanding, often requiring specialized depletion or capture methods that are difficult to optimize.

To overcome these challenges, we developed a pre-extraction protocol, designed to enrich samples for microbial DNA, by separating host cells from the associated microorganisms. Adapted and optimized from Bruggeling et al. (2021), our protocol consists into two phases: 1) cellular dissociation: microbial cells, host cells and sclerites (small calcareous structures embedded in the red coral tissue) are dissociated using a combination of enzymatic (collagenase IV or proteinase K) and mechanical digestion (mortar or ULTRA-TURRAX homogenizer) on tissue samples; 2) differential lysis: host cells are selectively lysed using the detergent Triton X-100, and the released host DNA is then degraded with DNase. Because bacterial cells remain intact during this step, they can subsequently be isolated and processed using a standard microbial DNA extraction protocol. qPCR analyses and metagenomic sequencing validated the efficacy of this approach, showing a significant enrichment of the microbial fraction: while standard extractions typically yield <2% of non-host sequences, our method achieved 30 to 58% of non-host sequences. This 15-to-30-fold increase in microbial signal significantly enhances the feasibility and cost effectiveness of MAG reconstruction from marine holobionts.