A framework for human microbiome research

A variety of microbial communities and their genes (the microbiome) exist throughout the human body, with fundamental roles in human health and disease. The National Institutes of Health (NIH)-funded Human Microbiome Project Consortium has established a population-scale framework to develop 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 or 18 body sites up to three times, which have generated 5,177 microbial taxonomic profiles from 16S ribosomal RNA genes and over 3.5 terabases of metagenomic sequence so far. In parallel, approximately 800 reference strains isolated from the human body have been sequenced. Collectively, these data represent the largest resource describing the abundance and variety of the human microbiome, while providing a framework for current and future studies

Structure, function and diversity of the healthy human microbiome

Author Posting. © The Authors, 2012. This article is posted here by permission of Nature Publishing Group. The definitive version was published in Nature 486 (2012): 207-214, doi:10.1038/nature11234.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 analysed the largest cohort and set of distinct, clinically relevant body habitats so far. 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.This research was supported in part by National Institutes of Health grants U54HG004969 to B.W.B.; U54HG003273 to R.A.G.; U54HG004973 to R.A.G., S.K.H. and J.F.P.; U54HG003067 to E.S.Lander; U54AI084844 to K.E.N.; N01AI30071 to R.L.Strausberg; U54HG004968 to G.M.W.; U01HG004866 to O.R.W.; U54HG003079 to R.K.W.; R01HG005969 to C.H.; R01HG004872 to R.K.; R01HG004885 to M.P.; R01HG005975 to P.D.S.; R01HG004908 to Y.Y.; R01HG004900 to M.K.Cho and P. Sankar; R01HG005171 to D.E.H.; R01HG004853 to A.L.M.; R01HG004856 to R.R.; R01HG004877 to R.R.S. and R.F.; R01HG005172 to P. Spicer.; R01HG004857 to M.P.; R01HG004906 to T.M.S.; R21HG005811 to E.A.V.; M.J.B. was supported by UH2AR057506; G.A.B. was supported by UH2AI083263 and UH3AI083263 (G.A.B., C. N. Cornelissen, L. K. Eaves and J. F. Strauss); S.M.H. was supported by UH3DK083993 (V. B. Young, E. B. Chang, F. Meyer, T. M. S., M. L. Sogin, J. M. Tiedje); K.P.R. was supported by UH2DK083990 (J. V.); J.A.S. and H.H.K. were supported by UH2AR057504 and UH3AR057504 (J.A.S.); DP2OD001500 to K.M.A.; N01HG62088 to the Coriell Institute for Medical Research; U01DE016937 to F.E.D.; S.K.H. was supported by RC1DE0202098 and R01DE021574 (S.K.H. and H. Li); J.I. was supported by R21CA139193 (J.I. and D. S. Michaud); K.P.L. was supported by P30DE020751 (D. J. Smith); Army Research Office grant W911NF-11-1-0473 to C.H.; National Science Foundation grants NSF DBI-1053486 to C.H. and NSF IIS-0812111 to M.P.; The Office of Science of the US Department of Energy under Contract No. DE-AC02-05CH11231 for P.S. C.; LANL Laboratory-Directed Research and Development grant 20100034DR and the US Defense Threat Reduction Agency grants B104153I and B084531I to P.S.C.; Research Foundation - Flanders (FWO) grant to K.F. and J.Raes; R.K. is an HHMI Early Career Scientist; Gordon&BettyMoore Foundation funding and institutional funding fromthe J. David Gladstone Institutes to K.S.P.; A.M.S. was supported by fellowships provided by the Rackham Graduate School and the NIH Molecular Mechanisms in Microbial Pathogenesis Training Grant T32AI007528; a Crohn’s and Colitis Foundation of Canada Grant in Aid of Research to E.A.V.; 2010 IBM Faculty Award to K.C.W.; analysis of the HMPdata was performed using National Energy Research Scientific Computing resources, the BluBioU Computational Resource at Rice University

EAnnot: A genome annotation tool using experimental evidence

The sequence of any genome becomes most useful for biological experimentation when a complete and accurate gene set is available. Gene prediction programs offer an efficient way to generate an automated gene set. Manual annotation, when performed by experienced annotators, is more accurate and complete than automated annotation. However, it is a laborious and expensive process, and by its nature, introduces a degree of variability not found with automated annotation. EAnnot (Electronic Annotation) is a program originally developed for manually annotating the human genome. It combines the latest bioinformatics tools to extract and analyze a wide range of publicly available data in order to achieve fast and reliable automatic gene prediction and annotation. EAnnot builds gene models based on mRNA, EST, and protein alignments to genomic sequence, attaches supporting evidence to the corresponding genes, identifies pseudogenes, and locates poly(A) sites and signals. Here, we compare manual annotation of human chromosome 6 with annotation performed by EAnnot in order to assess the latter's accuracy. EAnnot can readily be applied to manual annotation of other eukaryotic genomes and can be used to rapidly obtain an automated gene set

Genomes of Fasciola hepatica from the Americas Reveal Colonization with Neorickettsia Endobacteria Related to the Agents of Potomac Horse and Human Sennetsu Fevers

© 2017 McNulty et al. Food borne trematodes (FBTs) are an assemblage of platyhelminth parasites transmitted through the food chain, four of which are recognized as neglected tropical diseases (NTDs). Fascioliasis stands out among the other NTDs due to its broad and significant impact on both human and animal health, as Fasciola sp., are also considered major pathogens of domesticated ruminants. Here we present a reference genome sequence of the common liver fluke, Fasciola hepatica isolated from sheep, complementing previously reported isolate from cattle. A total of 14,642 genes were predicted from the 1.14 GB genome of the liver fluke. Comparative genomics indicated that F. hepatica Oregon and related food-borne trematodes are metabolically less constrained than schistosomes and cestodes, taking advantage of the richer millieux offered by the hepatobiliary organs. Protease families differentially expanded between diverse trematodes may facilitate migration and survival within the heterogeneous environments and niches within the mammalian host. Surprisingly, the sequencing of Oregon and Uruguay F. hepatica isolates led to the first discovery of an endobacteria in this species. Two contigs from the F. hepatica Oregon assembly were joined to complete the 859,205 bp genome of a novel Neorickettsia endobacterium (nFh) closely related to the etiological agents of human Sennetsu and Potomac horse fevers. Immunohistochemical studies targeting a Neorickettsia surface protein found nFh in specific organs and tissues of the adult trematode including the female reproductive tract, eggs, the Mehlis’ gland, seminal vesicle, and oral suckers, suggesting putative routes for fluke-to-fluke and fluke-to-host transmission. The genomes of F. hepatica and nFh will serve as a resource for further exploration of the biology of F. hepatica, and specifically its newly discovered trans-kingdom interaction with nFh and the impact of both species on disease in ruminants and humans

Genomes of <i>Fasciola hepatica</i> from the Americas Reveal Colonization with <i>Neorickettsia</i> Endobacteria Related to the Agents of Potomac Horse and Human Sennetsu Fevers

<div><p>Food borne trematodes (FBTs) are an assemblage of platyhelminth parasites transmitted through the food chain, four of which are recognized as neglected tropical diseases (NTDs). Fascioliasis stands out among the other NTDs due to its broad and significant impact on both human and animal health, as <i>Fasciola sp</i>., are also considered major pathogens of domesticated ruminants. Here we present a reference genome sequence of the common liver fluke, <i>Fasciola hepatica</i> isolated from sheep, complementing previously reported isolate from cattle. A total of 14,642 genes were predicted from the 1.14 GB genome of the liver fluke. Comparative genomics indicated that <i>F</i>. <i>hepatica</i> Oregon and related food-borne trematodes are metabolically less constrained than schistosomes and cestodes, taking advantage of the richer millieux offered by the hepatobiliary organs. Protease families differentially expanded between diverse trematodes may facilitate migration and survival within the heterogeneous environments and niches within the mammalian host. Surprisingly, the sequencing of Oregon and Uruguay <i>F</i>. <i>hepatica</i> isolates led to the first discovery of an endobacteria in this species. Two contigs from the <i>F</i>. <i>hepatica</i> Oregon assembly were joined to complete the 859,205 bp genome of a novel <i>Neorickettsia</i> endobacterium (<i>nFh</i>) closely related to the etiological agents of human Sennetsu and Potomac horse fevers. Immunohistochemical studies targeting a <i>Neorickettsia</i> surface protein found <i>nFh</i> in specific organs and tissues of the adult trematode including the female reproductive tract, eggs, the Mehlis’ gland, seminal vesicle, and oral suckers, suggesting putative routes for fluke-to-fluke and fluke-to-host transmission. The genomes of <i>F</i>. <i>hepatica</i> and <i>nFh</i> will serve as a resource for further exploration of the biology of <i>F</i>. <i>hepatica</i>, and specifically its newly discovered trans-kingdom interaction with <i>nFh</i> and the impact of both species on disease in ruminants and humans.</p></div

Phylogenetic affinities of the <i>Neorickettsia</i> symbiont of <i>Fasciola hepatica</i>.

<p>(A) The genomes of the four fully sequenced <i>Neorickettsia</i> species were aligned. Syntenic blocks are colored. The genome of the <i>Neorickettsia</i> of <i>Fasciola hepatica</i> (PRJNA295290) shares almost complete synteny with the genomes of <i>Neorickettsia risticii</i> (PRJNA19099) and <i>Neorickettsia sennetsu</i> (PRJNA357), with the exception of a small inversion that is also present in <i>Neorickettsia helminthoeca</i> (PRJNA187358). (B) Bayesian inference phylogenetic analysis based on available 16S ribosomal RNA sequences of <i>Neorickettsia</i> species, retrieved from [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006537#pgen.1006537.ref015" target="_blank">15</a>]. While the resolution of the sub-clade consisting of <i>Neorickettsia risticii</i> and <i>Neorickettsia</i> <i>sennetsu</i> is sub-optimal, the tree indicates that the <i>Neorickettsia</i> of <i>Fasciola hepatica</i> (<i>nFh</i>) may be most closely related to a strain that occurs in <i>Metagonimoides</i> species and an agent of Sennetsu fever. (C) A Bayesian inference phylogenetic tree based on the protein sequences of 473 single-copy, orthologous protein families conserved in the represented species clearly indicates that <i>nFh</i> is more closely related to <i>N</i>. <i>risticii</i> and <i>N</i>. <i>sennetsu</i> than <i>N</i>. <i>helminthoeca</i> or other species of the family Anaplasmataceae. NCBI GenBank accession numbers are indicated. Trematode hosts or other defining features are indicated for uncharacterized species.</p

Immunofluorescence detection of <i>Neorickettsia</i> in eggs of <i>F</i>. <i>hepatica</i> from Oregon using polyclonal anti-serum raised against a recombinant surface protein of Neorickettsia of <i>P</i>. <i>elegans</i> (PeNsp-3, green labeling, D-F).

<p>(A) Unstained eggs recovered from the liver by regular light microcopy. (B) and (C) Unstained eggs recovered from the liver by immunofluorescence microscopy using different filters demonstrating auto-fluorescence. (D-F) Cross-sections of eggs showing various amounts of <i>Neorickettsia</i> (arrows). Bar corresponds to 50 μm.</p

The sequenced genomes of <i>Neorickettsia</i> species.

<p>The sequenced genomes of <i>Neorickettsia</i> species.</p

The genome of the <i>Neorickettsia</i> endobacterium of <i>Fasciola hepatica</i>.

<p>The first track (from outside to inside) represents the 859,205bp genome of the <i>Neorickettsia</i> endobacterium of <i>Fasciola hepatica</i> (100 kb major ticks, 10 kb minor ticks). The genome shows nearly complete synteny with <i>Neorickettsia risticii</i> and <i>Neorickettsia sennetsu</i> with the exception of an inversion (shaded in grey). The second and third tracks represent the 744 inferred protein coding genes on the plus and minus strands, respectively. Genes are coded based on their NCBI Clusters of Orthologous Groups of proteins database classification. The fourth track represents RNA coding genes, including 3 ribosomal (red), 33 transfer (black), and 1 short, noncoding (blue). The fifth track depicts the G-C skew [(G-C)/(G+C)] calculated over 500bp windows.</p

Metabolic pathway reduction in parasitic flatworms.

<p>The global number of proteins assigned to different metabolic pathway was compared between different parasitic flatworms, the free-living planaria and cattle predicted proteomes. Those pathways showing a significant reduction in the parasitic species in relation to those present in planaria are indicated (strongly reduced: less than 30% conservation; reduced: conservation between 30 and 80%; not significantly reduced: more than 80% conservation). While in general several pathways are reduced in all parasitic species, some pathways are differential between food borne trematodes (<i>F</i>. <i>hepatica</i>, <i>O</i>. <i>viverrini</i>, <i>C</i>. <i>sinensis</i>), blood flukes (<i>S</i>. <i>mansoni</i> and <i>S</i>. <i>japonicum</i>) and cestodes (<i>E</i>. <i>multilocularis</i>, <i>H</i>. <i>microstoma</i> and <i>T</i>. <i>solium</i>). Most notably some lipid metabolism and amino acid pathways (i.e. aliphatic amino acid degradation) are not reduced in FBT (green) while they are reduced in the other groups. In general, FBT seem to be less constrained than blood flukes, with cestodes being the most restricted metabolically.</p