Studies of the human microbiome have revealed that even healthy individuals differ remarkably in the microbes that occupy habitats such as the gut, skin, and vagina. Much of this diversity remains unexplained, although diet, environment, host genetics, and early microbial exposure have all been implicated. Accordingly, to characterize the ecology of human-associated microbial communities, the Human Microbiome Project has analyzed the largest cohort and set of distinct, clinically relevant body habitats to date. We found the diversity and abundance of each habitat’s signature microbes to vary widely even among healthy subjects, with strong niche specialization both within and among individuals. The project encountered an estimated 81–99% of the genera, enzyme families, and community configurations occupied by the healthy Western microbiome. Metagenomic carriage of metabolic pathways was stable among individuals despite variation in community structure, and ethnic/racial background proved to be one of the strongest associations of both pathways and microbes with clinical metadata. These results thus delineate the range of structural and functional configurations normal in the microbial communities of a healthy population, enabling future characterization of the epidemiology, ecology, and translational applications of the human microbiome.

Bacterial diversity among environmental samples is commonly assessed with PCR-amplified 16S rRNA gene (16S) sequences. Perceived diversity, however, can be influenced by sample preparation, primer selection, and formation of chimeric 16S amplification products. Chimeras are hybrid products between multiple parent sequences that can be falsely interpreted as novel organisms, thus inflating apparent diversity. We developed a new chimera detection tool called Chimera Slayer (CS). CS detects chimeras with greater sensitivity than previous methods, performs well on short sequences such as those produced by the 454 Life Sciences (Roche) Genome Sequencer, and can scale to large data sets. By benchmarking CS performance against sequences derived from a controlled DNA mixture of known organisms and a simulated chimera set, we provide insights into the factors that affect chimera formation such as sequence abundance, the extent of similarity between 16S genes, and PCR conditions. Chimeras were found to reproducibly form among independent amplifications and contributed to false perceptions of sample diversity and the false identification of novel taxa, with less-abundant species exhibiting chimera rates exceeding 70%. Shotgun metagenomic sequences of our mock community appear to be devoid of 16S chimeras, supporting a role for shotgun metagenomics in validating novel organisms discovered in targeted sequence surveys.

A variety of microbial communities and their genes (microbiome) exist throughout the human body, playing fundamental roles in human health and disease. The NIH funded Human Microbiome Project (HMP) Consortium has established a population-scale framework which catalyzed significant development of metagenomic protocols resulting in a broad range of quality-controlled resources and data including standardized methods for creating, processing and interpreting distinct types of high-throughput metagenomic data available to the scientific community. Here we present resources from a population of 242 healthy adults sampled at 15 to 18 body sites up to three times, which to date, have generated 5,177 microbial taxonomic profiles from 16S rRNA genes and over 3.5 Tb of metagenomic sequence. In parallel, approximately 800 human-associated reference genomes have been sequenced. Collectively, these data represent the largest resource to date describing the abundance and variety of the human microbiome, while providing a platform for current and future studies.

Antibiotics administered in low doses have been widely used as growth promoters in the agricultural industry since the 1950s, yet the mechanisms for this effect are unclear. Because antimicrobial agents of different classes and varying activity are effective across several vertebrate species, we hypothesized that such subtherapeutic administration alters the population structure of the gut microbiome as well as its metabolic capabilities. We generated a model of adiposity by giving subtherapeutic antibiotic therapy to young mice and evaluated changes in the composition and capabilities of the gut microbiome. Administration of subtherapeutic antibiotic therapy increased adiposity in young mice and increased hormones related to metabolism. We observed substantial taxonomic changes in the microbiome, changes in copies of key genes involved in the metabolism of carbohydrates to short-chain fatty acids, increases in colonic short-chain fatty acid levels, and alterations in the regulation of hepatic metabolism of lipids and cholesterol. In this model, we demonstrate the alteration of early-life murine metabolic homeostasis through antibiotic manipulation.

Microbial communities carry out the majority of the biochemical activity on the planet, and they play integral roles in processes including metabolism and immune homeostasis in the human microbiome. Shotgun sequencing of such communities' metagenomes provides information complementary to organismal abundances from taxonomic markers, but the resulting data typically comprise short reads from hundreds of different organisms and are at best challenging to assemble comparably to single-organism genomes. Here, we describe an alternative approach to infer the functional and metabolic potential of a microbial community metagenome. We determined the gene families and pathways present or absent within a community, as well as their relative abundances, directly from short sequence reads. We validated this methodology using a collection of synthetic metagenomes, recovering the presence and abundance both of large pathways and of small functional modules with high accuracy. We subsequently applied this method, HUMAnN, to the microbial communities of 649 metagenomes drawn from seven primary body sites on 102 individuals as part of the Human Microbiome Project (HMP). This provided a means to compare functional diversity and organismal ecology in the human microbiome, and we determined a core of 24 ubiquitously present modules. Core pathways were often implemented by different enzyme families within different body sites, and 168 functional modules and 196 metabolic pathways varied in metagenomic abundance specifically to one or more niches within the microbiome. These included glycosaminoglycan degradation in the gut, as well as phosphate and amino acid transport linked to host phenotype (vaginal pH) in the posterior fornix. An implementation of our methodology is available at http://huttenhower.sph.harvard.edu/humann. This provides a means to accurately and efficiently characterize microbial metabolic pathways and functional modules directly from high-throughput sequencing reads, enabling the determination of community roles in the HMP cohort and in future metagenomic studies.