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Understanding the role of microbiota in maintaining health has been allowing for better disease prevention, diagnosis, and treatment. For example, healthiest gut microbiotas are resistant to invasion; conversely, gut microbiota vulnerability (that is the inverse of robustness) makes pathogenic invasion simpler. Thus, having a healthy, robust gut microbiota could help prevent pathogens from thriving. In other words, having a healthy, robust gut microbiota is desirable when antibiotic treatments are needed. Specific microbiota compositions are associated with better health conditions. Unravelling individual profiles, microbiota analysis helps promote a good health status and prevent intestinal or systemic diseases (including cancer) acting on microbiota composition.

Microbiota analysis can be useful to protect the health and well-being of and individual throughout life, from infancy to old age, and to develop individualized food plans and other lifestyle or, if needed, drugbased approaches aimed at correcting imbalances or improving microbiota composition. First studies on microbes – including those living in the gut – predominantly focused on individual species cultured in the laboratory. The first sequenced microbial genome (that of Haemophilus influenzae) was published in 1995. However, the vast majority of microbes – including bacteria living in the gut – cannot be cultured in the laboratory, and therefore cannot be studied with classical microbiological methods.

Moreover, culturing favors the selection of microbes best able to thrive under laboratory conditions, and not necessarily the dominant or the most influential one in the gut. Moreover, in nature, many microbes function as multicellular – and, often, multispecies – entities, interacting and communicating in complex ways. Today, we are in a new era, called metagenomics, in which the power of genomics (the study of the entire genetic material of an organism), bioinformatics, and system biology are combined to analyze the entire community of gut microbiota, bypassing individual microbial isolation and culture. Metagenomics transcends the individual microbe, focusing on genes and their reciprocal influence in the gut microbiota community.

New tools enable studying gut microbes in the complex community where they actually live, analyzing the genome of many microorganisms simultaneously. This allows understanding what gut microbiota are capable of, how they work, and the alterations that could lead to health problems. The first truly metagenomic survey of human gut microbiota appeared in 2006, and the first catalog of human microbiota bacterial genome, comprising 178 references, was published in 2010 by the Human Microbiome Project; until 2017, 437 gut microbiota genomes were sequenced. However, more than half of the sequences obtained from the analysis of a human gut microbiome cannot be mapped to existing bacterial reference genomes.

In 2019 a reference catalog of 1,520 nonredundant, high-quality draft bacterial genomes of human gut bacteria isolated using different culturing conditions (the Culturable Genome Reference) deposited in the China National GeneBank (CNGB) improved the mapping rate of selected metagenomics datasets to over 70%. The first step of a gut microbiota metagenomics study is DNA extraction from stool samples. The following DNA sequencing can capture a massive amount of information on gut microbiome. However, to study microbiota composition and diversity it is possible to focus on so-called ribosomal RNA (rRNA) phylotyping, a culture-independent method based on a database of more than 200,000 rRNA gene sequences.

rRNAs are essential components of cellular protein-making engines, ribosomes. All organisms, including bacteria, have rRNAs that are different enough to be distinguished one from another. Thus, the analysis of rRNA sequences in a stool sample allows for identifying gut microbiota composition. Focusing only on one gene, rRNA phylotyping represents a useful preliminary step to providing an assessment of gut microbiota diversity.