Targeted genomic characterization of uncultured bacteria from the human microbiot

来自人类微生物的未培养细菌的靶向基因组表征

• 批准号: 8325497
• 负责人: Mircea Podar
• 金额: $ 17.67万
• 依托单位: UT-BATTELLE, LLC-OAK RIDGE NATIONAL LAB
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-17 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8325497
• 关键词: Animals; Bacteria; Bacteroidetes; Biological Assay; Biological Preservation; Bypass; Categories; Cell Count; Cells; Characteristics; Chromosomes; Classification; Cloning; Communities; Complement; Complementarity Determining Regions; Complex; Computer Analysis; Cyanobacterium; DNA; DNA Sequence; Data; Detection; Development; Dideoxy Chain Termination DNA Sequencing; Experimental Designs; Facility Accesses; Family; Feasibility Studies; Flow Cytometry; Fluorescent in Situ Hybridization; Gene Amplification; Genetic; Genetic Drift; Genetic Population Study; Genome; Genomics; Goals; Health; Human; Human Microbiome; Human body; Individual; Label; Laboratories; Libraries; Mammalian Genetics; Metagenomics; Methods; Microbe; Microbiology; Molecular Biology; Monitor; Morphologic artifacts; Nucleic Acid Probes; Oligonucleotides; Organism; Pathogenesis; Phylogenetic Analysis; Physiological; Physiology; Population; Procedures; Proteins; Publishing; RNA Sequences; Radiation; Reaction; Research; Resolution; Ribosomal RNA; Sampling; Scientist; Selection Criteria; Shotgun Sequencing; Sorting - Cell Movement; Spatial Distribution; Species Specificity; Specificity; Speed; Systems Biology; Target Populations; Taxon; Techniques; Technology; Testing; Variant; Virulence; Whole Organism; base; cost; design; genome sequencing; gut microbiota; improved; microbial; microbial genome; microbiome; novel; pathogenic bacteria; physical conditioning; physical separation; rRNA Genes; scaffold; statistics; trait; vector; Animals; Bacteria; Bacteroidetes; Biological Assay; Biological Preservation; Bypass; Categories; Cell Count; Cells; Characteristics; Chromosomes; Classification; Cloning; Communities; Complement; Complementarity Determining Regions; Complex; Computer Analysis; Cyanobacterium; DNA; DNA Sequence; Data; Detection; Development; Dideoxy Chain Termination DNA Sequencing; Experimental Designs; Facility Accesses; Family; Feasibility Studies; Flow Cytometry; Fluorescent in Situ Hybridization; Gene Amplification; Genetic; Genetic Drift; Genetic Population Study; Genome; Genomics; Goals; Health; Human; Human Microbiome; Human body; Individual; Label; Laboratories; Libraries; Mammalian Genetics; Metagenomics; Methods; Microbe; Microbiology; Molecular Biology; Monitor; Morphologic artifacts; Nucleic Acid Probes; Oligonucleotides; Organism; Pathogenesis; Phylogenetic Analysis; Physiological; Physiology; Population; Procedures; Proteins; Publishing; RNA Sequences; Radiation; Reaction; Research; Resolution; Ribosomal RNA; Sampling; Scientist; Selection Criteria; Shotgun Sequencing; Sorting - Cell Movement; Spatial Distribution; Species Specificity; Specificity; Speed; Systems Biology; Target Populations; Taxon; Techniques; Technology; Testing; Variant; Virulence; Whole Organism; base; cost; design; genome sequencing; gut microbiota; improved; microbial; microbial genome; microbiome; novel; pathogenic bacteria; physical conditioning; physical separation; rRNA Genes; scaffold; statistics; trait; vector

DESCRIPTION (provided by applicant): A major goal of the Human Microbiome Project is to identify all of the organisms that are associated with the human body (the human microbiota) and determine the genomic sequence of most if not all of them. The detected diversity of the human microbiota reaches thousands of species and strains, the vast majority of which have not been isolated in pure culture. Our goal is to develop a robust and rapid approach for the targeted genomic characterization of any uncultured constituent of the human microbiota at single cell level and also to allow population genetic studies of selected groups of organisms that may have some cultured isolates. Our strategy utilizes the high phylogenetic resolution that the small subunit ribosomal RNA (SSU rRNA) provides in distinguishing microbial phylotypes. We plan to label and isolate single cells representing uncultured microbial lineages as well as populations of cells of specific phylotypes from complex microbiota samples and amplify their DNA to levels that enable genomic sequencing. This approach will bridge the gap between sequencing the limited number of individual cultured organisms and whole community shotgun sequencing (metagenomics) which generally does not provide sufficient depth and resolution to comprehensively sequence the microbiome. Initial feasibility studies indicate that our approach can be applied to any microbial consortia and is not dependent on the abundance of the target organism. Based on this, the focus of this proposal is to determine optimum experimental design and improved technical procedures for targeted single cell and population genomics of microbes from the human microbiota. The specific aims are to: 1. Aim 1. Separate single cells and populations of target uncultured microbial phylotypes from gut microbiota samples. We will use fluorescence in situ hybridization (FISH) combined with flow cytometry to obtain single cells and populations of targeted phylotypes, uncultured or with few cultured representatives 2. Aim 2. Amplification and sequencing of genomes from single cells representing the uncultured gut microbiota. We will amplify the genomes of target cells using multiple displacement amplification and sequence the DNA to obtain draft genomic assemblies. The experimental and computational approaches will be optimized for the human microbiota characteristics. 3. Aim 3. Pangenomic characterization of targeted populations of uncultured and cultured microbial phylotypes. We will isolate populations of specific bacterial phylotypes representing uncultured organisms as well as cell populations representing species/genera that have representatives in culture and one or few genomes sequenced. We will amplify and sequence the cell population genomic DNA to obtain composite genomes/pangenomes. PUBLIC HEALTH RELEVANCE: Targeted genomics of uncultured microbiota will enable selective access to the genetic blueprint of any of the organisms that inhabit the human body. This approach, which relies upon specific isolation of single cells or specific cell populations from complex human microbial consortia and sequencing their genomes or a part of, complements whole genome sequencing from individual cultivated organisms and global community metagenomics.

描述（由申请人提供）：人类微生物组项目的一个主要目标是识别与人体相关的所有生物（人类微生物群），并确定大多数（如果不是全部）的基因组序列。人类微生物群的多样性达到了数千种物种和菌株，其中绝大多数尚未在纯文化中孤立。我们的目标是为单个细胞水平的任何未培养的人类微生物群的靶向基因组表征开发出强大而快速的方法，并允许对可能具有某些培养的分离株的某些生物体的人群遗传研究。我们的策略利用了小型亚基核糖体RNA（SSU rRNA）提供的高系统发育分辨率，以区分微生物的系统型。我们计划标记和分离出代表未养殖的微生物谱系的单细胞以及从复杂的微生物群样品中特定系统型细胞的种群，并将其DNA放大至实现基因组测序的水平。这种方法将弥合排序有限数量的单个培养生物与整个社区shot弹枪测序（宏基因组学）之间的差距，这些生物通常无法提供足够的深度和解决方案来全面地对微生物组进行序列。最初的可行性研究表明，我们的方法可以应用于任何微生物群体，并且不依赖于目标生物的丰度。基于此，该提案的重点是确定人类微生物群体微生物的靶向单细胞和种群基因组学的最佳实验设计和改进的技术程序。具体目的是：1。AIM1。与肠道微生物群样品的单独的单细胞和目标未培养的微生物系统型的种群。我们将使用荧光原位杂交（FISH）与流式细胞术结合，以获得靶向系统型的单细胞和群体，未经培养或培养的代表很少。目标2。AIM 2。从代表未养殖的肠菌Microbobiota的单个细胞的扩增和测序基因组。我们将使用多个位移扩增扩大靶细胞的基因组，并对DNA进行序列以获得基因组组件草案。实验和计算方法将针对人类微生物群特征进行优化。 3。目标3。未经培养和培养的微生物系统型的靶向种群的pangenomic表征。我们将分离代表未培养的生物的特定细菌系统型的种群以及代表具有培养物代表和一个或几个基因组的物种/属的细胞群体。我们将放大并对细胞群基因组DNA进行测序，以获得复合基因组/pangenomes。公共卫生相关性：未培养的微生物群的靶向基因组学将使人们能够选择性地访问居住在人体的任何生物的遗传蓝图。这种方法依赖于从复杂的人类微生物consortia中的单细胞或特定细胞群体的特定分离，并对其基因组进行测序或一部分，它补充了来自单个培养生物体和全球社区元基因组学的整个基因组测序。