Holo-2bRAD: A Hologenomic Method for High-Resolution Analysis of Coral Microbiomes During Bleaching

Coral reefs are biodiversity hotspots increasingly threatened by climate-induced bleaching, yet profiling the coral holobiont-the host and its associated microbiota-remains technically challenging due to high host-DNA contamination (often >95%) and the lack of comprehensive reference databases. Here, we present holo-2bRAD, a type IIB restriction site-associated DNA sequencing approach. This method, strategically integrated with a meticulously curated hologenome database (comprising 404,946 microbial genomes and 56 coral-derived metagenome-assembled genomes), effectively overcomes overwhelming host contamination (~99%). We demonstrate its exceptional species specificity (99.92%) in profiling Galaxea fascicularis (Linnaeus, 1767; Order Scleractinia, Family Euphylliidae) holobionts across bleaching severities, thereby validating its technical feasibility. Leveraging this high-resolution tool, our hologenome analysis revealed significant restructuring of coral-associated microbiota during bleaching, where microbial shifts (e.g., depletion of beneficial Thermoanaerobacterium thermosaccharolyticum and enrichment of stress-responsive bacteria) correlated more strongly with bleaching phenotypes than host genetic variation. By providing cost-effective, multi-domain hologenome profiling at unprecedented resolution, holo-2bRAD offers a practical tool for investigating holobiont dynamics and developing microbiome-informed coral conservation strategies.