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Metagenomics has revolutionized our understanding of microbial communities, offering unprecedented insights into their genetic and functional diversity across Earth’s diverse ecosystems. Beyond their roles as environmental constituents, microbiomes act as symbionts, profoundly influencing the health and function of their host organisms. Given the inherent complexity of these communities and the diverse environments where they reside, the components of a metagenomics study must be carefully tailored to yield accurate results that are representative of the populations of interest. This Primer examines the methodological advancements and current practices that have shaped the field, from initial stages of sample collection and DNA extraction to the advanced bioinformatics tools employed for data analysis, with a particular focus on the profound impact of next-generation sequencing on the scale and accuracy of metagenomics studies. We critically assess the challenges and limitations inherent in metagenomics experimentation, available technologies and computational analysis methods. Beyond technical methodologies, we explore the application of metagenomics across various domains, including human health, agriculture and environmental monitoring. Looking ahead, we advocate for the development of more robust computational frameworks and enhanced interdisciplinary collaborations. This Primer serves as a comprehensive guide for advancing the precision and applicability of metagenomic studies, positioning them to address the complexities of microbial ecology and their broader implications for human health and environmental sustainability. 

Abstract Stony coral tissue loss disease (SCTLD) is a widespread and deadly disease that affects nearly half of Caribbean coral species. To understand the microbial community response to this disease, we performed a disease transmission experiment on US Virgin Island (USVI) corals, exposing six species of coral with varying susceptibility to SCTLD. The microbial community of the surface mucus and tissue layers were examined separately using a small subunit ribosomal RNA gene-based sequencing approach, and data were analyzed to identify microbial community shifts following disease acquisition, potential causative pathogens, as well as compare microbiota composition to field-based corals from the USVI and Florida outbreaks. While all species displayed similar microbiome composition with disease acquisition, microbiome similarity patterns differed by both species and mucus or tissue microhabitat. Further, disease exposed but not lesioned corals harbored a mucus microbial community similar to those showing disease signs, suggesting that mucus may serve as an early warning detection for the onset of SCTLD. Like other SCTLD studies in Florida, Rhodobacteraceae, Arcobacteraceae, Desulfovibrionaceae, Peptostreptococcaceae, Fusibacter, Marinifilaceae, and Vibrionaceae dominated diseased corals. This study demonstrates the differential response of the mucus and tissue microorganisms to SCTLD and suggests that mucus microorganisms may be diagnostic for early disease exposure. 

Stony coral tissue loss disease (SCTLD) was initially documented in Florida in 2014 and outbreaks with similar characteristics have since appeared in disparate areas throughout the northern Caribbean, causing significant declines in coral communities. SCTLD is characterized by focal or multifocal lesions of denuded skeleton caused by rapid tissue loss and affects at least 22 reef-building species of Caribbean corals. A tissue-loss disease consistent with the case definition of SCTLD was first observed in the U.S. Virgin Islands (USVI) in January of 2019 off the south shore of St. Thomas at Flat Cay. The objective of the present study was to characterize species susceptibility to the disease present in St. Thomas in a controlled laboratory transmission experiment. Fragments of six species of corals ( Colpophyllia natans , Montastraea cavernosa , Orbicella annularis , Porites astreoides , Pseudodiploria strigosa , and Siderastrea siderea ) were simultaneously incubated with (but did not physically contact) SCTLD-affected colonies of Diploria labyrinthiformis and monitored for lesion appearance over an 8 day experimental period. Paired fragments from each corresponding coral genotype were equivalently exposed to apparently healthy colonies of D. labyrinthiformis to serve as controls; none of these fragments developed lesions throughout the experiment. When tissue-loss lesions appeared and progressed in a disease treatment, the affected coral fragment, and its corresponding control genet, were removed and preserved for future analysis. Based on measures including disease prevalence and incidence, relative risk of lesion development, and lesion progression rates, O. annularis, C. natans , and S. siderea showed the greatest susceptibility to SCTLD in the USVI. These species exhibited earlier average development of lesions, higher relative risk of lesion development, greater lesion prevalence, and faster lesion progression rates compared with the other species, some of which are considered to be more susceptible based on field observations (e.g., P. strigosa ). The average transmission rate in the present study was comparable to tank studies in Florida, even though disease donor species differed. Our findings suggest that the tissue loss disease affecting reefs of the USVI has a similar epizootiology to that observed in other regions, particularly Florida.