We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view of chromatin structure has emerged, including its inter-relationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded new mechanistic and evolutionary insights concerning the functional landscape of the human genome. Together, these studies are defining a path for pursuit of a more comprehensive characterization of human genome function.

Human skin is a large, heterogeneous organ that protects the body from pathogens while sustaining microorganisms that influence human health and disease. Our analysis of 16S ribosomal RNA gene sequences obtained from 20 distinct skin sites of healthy humans revealed that physiologically comparable sites harbor similar bacterial communities. The complexity and stability of the microbial community are dependent on the specific characteristics of the skin site. This topographical and temporal survey provides a baseline for studies that examine the role of bacterial communities in disease states and the microbial interdependencies required to maintain healthy skin.The skin is a critical interface between the human body and its external environment, preventing loss of moisture and barring entry of pathogenic organisms (1). The skin is also an ecosystem, harboring microbial communities that live in a range of physiologically and topographically distinct niches (2). For example, hairy, moist underarms lie a short distance from smooth dry forearms, but these two niches are likely as ecologically dissimilar as rainforests are to deserts. Traditional culture-based characterizations of the skin microbiota are biased toward species that readily grow under standard laboratory conditions, such as Staphylococci spp. However, †To whom correspondence should be addressed. jsegre@nhgri.nih.gov. * See supporting online material for names of group members. Characterizing the microbiota that inhabit specific sites may provide insight into the delicate balance between skin health and disease. Certain dermatological disorders manifest at stereotypical skin sites [e.g., psoriasis on the outer elbow and atopic dermatitis (eczema) on the inner bend of the elbow]. Moreover, antibiotic exposure, modified hygienic practices, and lifestyle changes have the potential to alter the skin microbiome selectively and may underlie the increased incidence of human disorders such as atopic dermatitis. Understanding naturally occurring symbiotic microbial communities will provide insight into the conditions that favor the emergence of antibiotic-resistant organisms [e.g., the highly pathogenic strain of methicillin-resistant S. aureus, which acquired genes that promote growth on skin from the symbiont S. epidermidis (6)].We characterized the topographical and temporal diversity of the human skin microbiome with the use of 16S rRNA gene phylotyping, and generated 112,283 near-full-length bacterial 16S gene sequences from samples of 20 diverse skin sites on each of 10 healthy humans (7) (fig. S1 and table S1). Nineteen bacterial phyla were detected, but most sequences were assigned to four phyla: Actinobacteria (51.8%), Firmicutes (24.4%), Proteobacteria (16.5%), and Bacteroidetes (6.3%). Of the 205 identified genera represented by at least five sequences, three were associated with more than 62% of the sequences: Corynebacteria (22.8%; Actinobacteria), Propionibacteria (23.0%; Actinobacteria), and Staphylococci (16.8%; Firmicutes). At the species...

The many layers and structures of the skin serve as elaborate hosts to microbes, including a diversity of commensal and pathogenic bacteria that contribute to both human health and disease. To determine the complexity and identity of the microbes inhabiting the skin, we sequenced bacterial 16S small-subunit ribosomal RNA genes isolated from the inner elbow of five healthy human subjects. This analysis revealed 113 operational taxonomic units (OTUs; “phylotypes”) at the level of 97% similarity that belong to six bacterial divisions. To survey all depths of the skin, we sampled using three methods: swab, scrape, and punch biopsy. Proteobacteria dominated the skin microbiota at all depths of sampling. Interpersonal variation is approximately equal to intrapersonal variation when considering bacterial community membership and structure. Finally, we report strong similarities in the complexity and identity of mouse and human skin microbiota. This study of healthy human skin microbiota will serve to direct future research addressing the role of skin microbiota in health and disease, and metagenomic projects addressing the complex physiological interactions between the skin and the microbes that inhabit this environment.

Chromatin-based functional genomic analyses and genomewide association studies (GWASs) together implicate enhancers as critical elements influencing gene expression and risk for common diseases. Here, we performed systematic chromatin and transcriptome profiling in human pancreatic islets. Integrated analysis of islet data with those from nine cell types identified specific and significant enrichment of type 2 diabetes and related quantitative trait GWAS variants in islet enhancers. Our integrated chromatin maps reveal that most enhancers are short (median = 0.8 kb). Each cell type also contains a substantial number of more extended (≥3 kb) enhancers. Interestingly, these stretch enhancers are often tissue-specific and overlap locus control regions, suggesting that they are important chromatin regulatory beacons. Indeed, we show that (i) tissue specificity of enhancers and nearby gene expression increase with enhancer length; (ii) neighborhoods containing stretch enhancers are enriched for important cell type-specific genes; and (iii) GWAS variants associated with traits relevant to a particular cell type are more enriched in stretch enhancers compared with short enhancers. Reporter constructs containing stretch enhancer sequences exhibited tissue-specific activity in cell culture experiments and in transgenic mice. These results suggest that stretch enhancers are critical chromatin elements for coordinating cell type-specific regulatory programs and that sequence variation in stretch enhancers affects risk of major common human diseases.

The systematic comparison of genomic sequences from different organisms represents a central focus of contemporary genome analysis. Comparative analyses of vertebrate sequences can identify coding and conserved non-coding regions, including regulatory elements, and provide insight into the forces that have rendered modern-day genomes. As a complement to whole-genome sequencing efforts, we are sequencing and comparing targeted genomic regions in multiple, evolutionarily diverse vertebrates. Here we report the generation and analysis of over 12 megabases (Mb) of sequence from 12 species, all derived from the genomic region orthologous to a segment of about 1.8 Mb on human chromosome 7 containing ten genes, including the gene mutated in cystic fibrosis. These sequences show conservation reflecting both functional constraints and the neutral mutational events that shaped this genomic region. In particular, we identify substantial numbers of conserved non-coding segments beyond those previously identified experimentally, most of which are not detectable by pair-wise sequence comparisons alone. Analysis of transposable element insertions highlights the variation in genome dynamics among these species and confirms the placement of rodents as a sister group to the primates.

The laboratory rat (Rattus norvegicus) is an indispensable tool in experimental medicine and drug development, having made inestimable contributions to human health. We report here the genome sequence of the Brown Norway (BN) rat strain. The sequence represents a high-quality 'draft' covering over 90% of the genome. The BN rat sequence is the third complete mammalian genome to be deciphered, and three-way comparisons with the human and mouse genomes resolve details of mammalian evolution. This first comprehensive analysis includes genes and proteins and their relation to human disease, repeated sequences, comparative genome-wide studies of mammalian orthologous chromosomal regions and rearrangement breakpoints, reconstruction of ancestral karyotypes and the events leading to existing species, rates of variation, and lineage-specific and lineage-independent evolutionary events such as expansion of gene families, orthology relations and protein evolution.

Public health officials have raised concerns that plasmid transfer between Enterobacteriaceae species may spread resistance to carbapenems, an antibiotic class of last resort, thereby rendering common healthcare-associated infections nearly impossible to treat. We performed comprehensive surveillance and genomic sequencing to identify carbapenem-resistant Enterobacteriaceae in the NIH Clinical Center patient population and hospital environment in order to to articulate the diversity of carbapenemase-encoding plasmids and survey the mobility of and assess the mobility of these plasmids between bacterial species. We isolated a repertoire of carbapenemase-encoding Enterobacteriaceae, including multiple strains of Klebsiella pneumoniae, Klebsiella oxytoca, Escherichia coli, Enterobacter cloacae, Citrobacter freundii, and Pantoea species. Long-read genome sequencing with full end-to-end assembly revealed that these organisms carry the carbapenem-resistance genes on a wide array of plasmids. Klebsiella pneumoniae and Enterobacter cloacae isolated simultaneously from a single patient harbored two different carbapenemase-encoding plasmids, overriding the epidemiological scenario of plasmid transfer between organisms within this patient. We did, however, find evidence supporting horizontal transfer of carbapenemase-encoding plasmids between Klebsiella pneumoniae, Enterobacter cloacae and Citrobacter freundii in the hospital environment. Our comprehensive sequence data, with full plasmid identification, challenges assumptions about horizontal gene transfer events within patients and identified wider possible connections between patients and the hospital environment. In addition, we identified a new carbapenemase-encoding plasmid of potentially high clinical impact carried by Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae and Pantoea species, from unrelated patients and the hospital environment.

HIV-1-neutralizing antibodies develop in most HIV-1-infected individuals, although highly effective antibodies are generally observed only after years of chronic infection. Here we characterize the rate of maturation and extent of diversity for the lineage that produced the broadly neutralizing antibody VRC01 through longitudinal sampling of peripheral B cell transcripts over 15 years and co-crystal structures of lineage members. Next-generation sequencing identified VRC01-lineage transcripts, which encompassed diverse antibodies organized into distinct phylogenetic clades. Prevalent clades maintained characteristic features of antigen recognition, though each evolved binding loops and disulfides that formed distinct recognition surfaces. Over the course of the study period, VRC01-lineage clades showed continuous evolution, with rates of ~2 substitutions per 100 nucleotides per year, comparable to that of HIV-1 evolution. This high rate of antibody evolution provides a mechanism by which antibody lineages can achieve extraordinary diversity and, over years of chronic infection, develop effective HIV-1 neutralization.