The gut microbiota is a critical player in human health and disease by shaping human immunity and metabolism. Studies, including ours, have demonstrated links between gut microbiota alterations and HIV-1 immunopathogenesis, as well as chronic clinical complications, e.g., cardiovascular diseases. However, there are still substantial knowledge gaps. The microbiota contains a wide variety of microorganisms (bacteria, viruses, archaea, and fungi). The term microbiome refers to the entire habitat and their genes/genomes. Previous microbiome studies were based on either 16S rRNA gene sequencing which only profiles bacterial composition with low-resolution limited to genus level at best, or reference-based metagenomics which relies on reference sequences of known taxa and genes in the microbiome. Consequently, the so far reported HIV-related microbiome biomarkers are limited to known bacteria which constitute less than half of gut microbiota. Our pilot study found that using shotgun metagenome sequencing in people living with HIV (PLWH), most metagenome sequences (60%) were unmapped to any known microbial genomes. We hypothesize that these unknown/uncultured microbes play important roles in regulating physiology and immunity in PLWH. Our hypothesis is supported by a recent microbiome study in mice which applied a novel metagenomics approach integrating genomes of known and uncultured microbes showing that the strongest drivers of the diet-induced changes in mice were previously undetected taxa. The HIV-related gut microbiome signatures are thus to be redefined. In addition, the other microbial kingdoms, viruses (virome) and fungi (mycobiome), are understudied. Although their abundance in the human gut microbiome is much lower compared with bacteria, also shown by our pilot study, accumulating evidence suggests that they regulate physiology via multiple interactions with host cells within the gastrointestinal tract. The role of these other kingdoms of the microbiome in PLWH is yet to be clarified. In this project, we will analyze a large amount of metagenome data from very well-defined HIV cohorts using the novel metagenomics pipeline MetaPhlAn 4 to characterize the bacterial microbiome (bacteriome) and redefine the HIV-related bacteriome signatures. MetaPhlAn 4 uses the Species-level Genome Bin strategy to group both reference genomes and metagenome-assembled genomes (MAGs) into known and unknown species. An unknown species is a proxy for a species that remains uncultivated and whose existence relies on information from MAGs. In addition, we will establish new pipelines to characterize gut virome and mycobiome in relation to HIV infection and patient outcomes. Ultimately, by applying advanced bioinformatics and machine learning, new microbial species and metabolic pathways related to HIV infection and host immunity will be discovered. The study of the associations between virome and bacteriome biomarkers and clinical risk of e.g. cardiovascular events in this patient population offers the possibility to progress such biomarkers into clinical validation, by establishing standardized protocols that could also be exploited in different health conditions in non-HIV infected populations. Given the existence of microbiome-driven therapeutic approaches, the findings will offer an exciting prospect for personalized medicine, both early disease diagnosis and personalized therapies.