Large genome databases have markedly improved our understanding of marine microorganisms1–5. Although these resources have focused on prokaryotes, genomes from many dominant marine lineages, such as Pelagibacter and Prochlorococcus, are conspicuously underrepresented. Here we present the Great Barrier Reef Microbial Genomes Database (GBR-MGD), comprising 5,283 prokaryotic genomes obtained from Great Barrier Reef seawater samples using Nanopore and Illumina sequencing, including a collection of high-quality genomes of underrepresented groups. We show that standard short-read assemblies miss these populations owing to a combination of strain heterogeneity and low-GC-percentage sequencing bias. The GBR-MGD also comprises 20 chromosome-level picoeukaryote and 808,585 viral genomes, including a newly described clade of marine Crassvirales. We demonstrate the utility of the GBR-MGD to identify indicator taxa that can reliably predict the effects of reef management practices, such as the establishment of marine protected zones.
