Chapter 12: Human Microbiome Analysis

Humans are essentially sterile during gestation, but during and after birth, every body surface, including the skin, mouth, and gut, becomes host to an enormous variety of microbes, bacterial, archaeal, fungal, and viral. Under normal circumstances, these microbes help us to digest our food and to maintain our immune systems, but dysfunction of the human microbiota has been linked to conditions ranging from inflammatory bowel disease to antibiotic-resistant infections. Modern high-throughput sequencing and bioinformatic tools provide a powerful means of understanding the contribution of the human microbiome to health and its potential as a target for therapeutic interventions. This chapter will first discuss the historical origins of microbiome studies and methods for determining the ecological diversity of a microbial community. Next, it will introduce shotgun sequencing technologies such as metagenomics and metatranscriptomics, the computational challenges and methods associated with these data, and how they enable microbiome analysis. Finally, it will conclude with examples of the functional genomics of the human microbiome and its influences upon health and disease

PhyloPhlAn is a new method for improved phylogenetic and taxonomic placement of microbes

New microbial genomes are constantly being sequenced, and it is crucial to accurately determine their taxonomic identities and evolutionary relationships. Here we report PhyloPhlAn, a new method to assign microbial phylogeny and putative taxonomy using >400 proteins optimized from among 3,737 genomes. This method measures the sequence diversity of all clades, classifies genomes from deep-branching candidate divisions through closely-related subspecies, and improves consistency between phylogenetic and taxonomic groupings. PhyloPhlAn improved taxonomic accuracy for existing and newly-sequenced genomes, detecting 157 erroneous labels, correcting 46, and placing or refining 130 new genomes. We provide examples of accurate classifications from subspecies (Sulfolobus spp.) to phyla, and of preliminary rooting of deep-branching candidate divisions, including consistent statistical support for Caldiserica (formerly candidate division OP5). PhyloPhlAn will thus be useful for both phylogenetic assessment and taxonomic quality control of newly-sequenced genomes. The final phylogenies, conserved protein sequences, and open-source implementation are available online

Computational meta'omics for microbial community studies

Complex microbial communities are an integral part of the Earth's ecosystem and of our bodies in health and disease. In the last two decades, culture-independent approaches have provided new insights into their structure and function, with the exponentially decreasing cost of high-throughput sequencing resulting in broadly available tools for microbial surveys. However, the field remains far from reaching a technological plateau, as both computational techniques and nucleotide sequencing platforms for microbial genomic and transcriptional content continue to improve. Current microbiome analyses are thus starting to adopt multiple and complementary meta'omic approaches, leading to unprecedented opportunities to comprehensively and accurately characterize microbial communities and their interactions with their environments and hosts. This diversity of available assays, analysis methods, and public data is in turn beginning to enable microbiome-based predictive and modeling tools. We thus review here the technological and computational meta'omics approaches that are already available, those that are under active development, their success in biological discovery, and several outstanding challenges

Advancing the Microbiome Research Community

The human microbiome has become a recognized factor in promoting and maintaining health. We outline opportunities in interdisciplinary research, analytical rigor, standardization, and policy development for this relatively new and rapidly developing field. Advances in these aspects of the research community may in turn advance our understanding of human microbiome biology. It is now widely recognized that disturbances in our normal microbial populations may be linked to acute infections such as Clostridium difficile and to chronic diseases such as heart disease, cancer, obesity, and autoimmune disorders (Clemente et al., 2012). This has prompted substantial interest in the microbiome from both basic and clinical perspectives. Although our genome is relatively static throughout life, each of our microbial communities changes profoundly from infancy through adulthood, continuing to adapt through ongoing exposures to diet, drugs and environment. Understanding the microbiome and its dynamic nature may be critical for diagnostics and, eventually, interventions based on the microbiome itself. However, several important challenges limit the ability of researchers to enter the microbiome field and/or conduct research most effectively

Reprograming of gut microbiome energy metabolism by the FUT2 Crohn's disease risk polymorphism.

Fucosyltransferase 2 (FUT2) is an enzyme that is responsible for the synthesis of the H antigen in body fluids and on the intestinal mucosa. The H antigen is an oligosaccharide moiety that acts as both an attachment site and carbon source for intestinal bacteria. Non-secretors, who are homozygous for the loss-of-function alleles of FUT2 gene (sese), have increased susceptibility to Crohn's disease (CD). To characterize the effect of FUT2 polymorphism on the mucosal ecosystem, we profiled the microbiome, meta-proteome and meta-metabolome of 75 endoscopic lavage samples from the cecum and sigmoid of 39 healthy subjects (12 SeSe, 18 Sese and 9 sese). Imputed metagenomic analysis revealed perturbations of energy metabolism in the microbiome of non-secretor and heterozygote individuals, notably the enrichment of carbohydrate and lipid metabolism, cofactor and vitamin metabolism and glycan biosynthesis and metabolism-related pathways, and the depletion of amino-acid biosynthesis and metabolism. Similar changes were observed in mice bearing the FUT2(-/-) genotype. Metabolomic analysis of human specimens revealed concordant as well as novel changes in the levels of several metabolites. Human metaproteomic analysis indicated that these functional changes were accompanied by sub-clinical levels of inflammation in the local intestinal mucosa. Therefore, the colonic microbiota of non-secretors is altered at both the compositional and functional levels, affecting the host mucosal state and potentially explaining the association of FUT2 genotype and CD susceptibility

Relating the metatranscriptome and metagenome of the human gut

Although the composition of the human microbiome is now wellstudied, the microbiota’s \u3e8 million genes and their regulation remain largely uncharacterized. This knowledge gap is in part because of the difficulty of acquiring large numbers of samples amenable to functional studies of the microbiota. We conducted what is, to our knowledge, one of the first human microbiome studies in a well-phenotyped prospective cohort incorporating taxonomic, metagenomic, and metatranscriptomic profiling at multiple body sites using self-collected samples. Stool and saliva were provided by eight healthy subjects, with the former preserved by three different methods (freezing, ethanol, and RNAlater) to validate self-collection. Within-subject microbial species, gene, and transcript abundances were highly concordant across sampling methods, with only a small fraction of transcripts (\u3c5%) displaying between-method variation. Next, we investigated relationships between the oral and gut microbial communities, identifying a subset of abundant oral microbes that routinely survive transit to the gut, but with minimal transcriptional activity there. Finally, systematic comparison of the gut metagenome and metatranscriptome revealed that a substantial fraction (41%) of microbial transcripts were not differentially regulated relative to their genomic abundances. Of the remainder, consistently underexpressed pathways included sporulation and amino acid biosynthesis, whereas up-regulated pathways included ribosome biogenesis and methanogenesis. Across subjects, metatranscriptional profiles were significantly more individualized than DNA-level functional profiles, but less variable than microbial composition, indicative of subject-specific whole-community regulation. The results thus detail relationships between community genomic potential and gene expression in the gut, and establish the feasibility of metatranscriptomic investigations in subject-collected and shipped samples

Functional profiling of the gut microbiome in disease-associated inflammation

The microbial residents of the human gut are a major factor in the development and lifelong maintenance of health. The gut microbiota differs to a large degree from person to person and has an important influence on health and disease due to its interaction with the human immune system. Its overall composition and microbial ecology have been implicated in many autoimmune diseases, and it represents a particularly important area for translational research as a new target for diagnostics and therapeutics in complex inflammatory conditions. Determining the biomolecular mechanisms by which altered microbial communities contribute to human disease will be an important outcome of current functional studies of the human microbiome. In this review, we discuss functional profiling of the human microbiome using metagenomic and metatranscriptomic approaches, focusing on the implications for inflammatory conditions such as inflammatory bowel disease and rheumatoid arthritis. Common themes in gut microbial ecology have emerged among these diverse diseases, but they have not yet been linked to targetable mechanisms such as microbial gene and genome composition, pathway and transcript activity, and metabolism. Combining these microbial activities with host gene, transcript and metabolic information will be necessary to understand how and why these complex interacting systems are altered in disease-associated inflammation

Assessing the functional structure of genomic data

Motivation: The availability of genome-scale data has enabled an abundance of novel analysis techniques for investigating a variety of systems-level biological relationships. As thousands of such datasets become available, they provide an opportunity to study high-level associations between cellular pathways and processes. This also allows the exploration of shared functional enrichments between diverse biological datasets, and it serves to direct experimenters to areas of low data coverage or with high probability of new discoveries

The microbiome quality control project: baseline study design and future directions

Microbiome research has grown exponentially over the past several years, but studies have been difficult to reproduce across investigations. Relevant variation in measurements between laboratories, from a variety of sources, has not been systematically assessed. This is coupled with a growing concern in the scientific community about the lack of reproducibility in biomedical research. The Microbiome Quality Control project (MBQC) was initiated to identify sources of variation in microbiome studies, to quantify their magnitudes, and to assess the design and utility of different positive and negative control strategies. Here we report on the first MBQC baseline study project and workshop

Analysis of 1321 Eubacterium rectale genomes from metagenomes uncovers complex phylogeographic population structure and subspecies functional adaptations

Funding This work was supported by NIH NHGRI grant R01HG005220, NIDDK grant R24DK110499, NIDDK grant U54DE023798, and CMIT grant 6935956 to C.H., and by the European Research Council (ERC-STG project MetaPG-716575), MIUR “Futuro in Ricerca” RBFR13EWWI_001, the European Union (H2020-SFS-2018-1 project MASTER-818368 and H2020-SC1-BHC project ONCOBIOME-825410), and the National Cancer Institute of the National Institutes of Health (1U01CA230551) to N.S. Further support was provided by the Programma Ricerca Budget prestazioni Eurac 2017 of the Province of Bolzano, Italy to F.M., and by the EU-H2020 (DiMeTrack-707345) to E.P. and N.S. D.B., S.H.D., P.L., A.W.W. and The Rowett Institute received core funding support from the Scottish Government Rural and Environmental Sciences and Analytical Services (SG-RESAS). Availability of data and materials All datasets used in this study are publicly available and matched with their respective PMID (Additional file 5). The high-quality E. rectale MAGs in fasta format and a metadata file are available at http://segatalab.cibio.unitn.it/data/Erectale_Karcher_et_al.html and in the following Zenodo repository: https://doi.org/10.5281/zenodo.3763191 [80]. The two new isolate genomes L2–21 and T3BWe13 have been uploaded to NCBI and can be found in RefSeq under the accession numbers GCF_008122485.1 [81] and GCF_008123415.1 [82], respectively.Peer reviewedPublisher PD