Targeted genomic characterization of uncultured bacteria from the human microbiot

描述(由申请人提供)：人类微生物组计划的一个主要目标是识别与人体有关的所有生物(人类微生物区系)，并确定大多数(如果不是全部)的基因组序列。检测到的人类微生物区系的多样性达到了数千个物种和菌株，其中绝大多数没有在纯培养中分离出来。我们的目标是开发一种强大而快速的方法，在单细胞水平上对人类微生物区系中任何未培养的组成部分进行有针对性的基因组特征描述，并允许对可能具有某些培养分离株的选定生物群进行群体遗传学研究。我们的策略利用小亚单位核糖体RNA(SSU RRNA)在区分微生物系统类型方面提供的高系统发育分辨率。我们计划标记和分离代表未培养微生物谱系的单个细胞，以及从复杂微生物群样本中分离出特定种型的细胞群，并将它们的DNA扩增到能够进行基因组测序的水平。这种方法将弥合有限数量的单个培养生物体测序和整个社区鸟枪测序(元基因组学)之间的差距，后者通常不能提供足够的深度和分辨率来全面测序微生物组。初步可行性研究表明，我们的方法可以应用于任何微生物联合体，而不依赖于目标生物体的丰度。基于此，这项提议的重点是确定最优的实验设计和改进的技术程序，用于从人类微生物群中定向单细胞和群体基因组学。具体目的是：1.目的1.从肠道微生物区系样品中分离出目标未培养微生物系统型的单个细胞和种群。我们将使用荧光原位杂交(FISH)和流式细胞术相结合的方法来获得目标系统类型的单个细胞和群体，包括未培养的或几乎没有培养的代表性的。2.目的2.从代表未培养的肠道微生物区系的单个细胞中扩增基因组并测序。我们将使用多次置换扩增来扩增靶细胞的基因组，并对DNA进行测序以获得草稿基因组组装。实验和计算方法将针对人类微生物区系的特点进行优化。3.目的3.未培养和培养微生物系统型目标种群的基因组学特征。我们将分离代表未培养生物的特定细菌系统型的种群，以及代表在培养中具有代表性的种/属的细胞种群，并对一个或几个基因组进行测序。我们将对细胞群体基因组DNA进行扩增和测序，以获得复合基因组/泛基因组。与公共卫生相关：对未培养的微生物区系进行定向基因组学研究，将使人们能够有选择地获得居住在人体内的任何生物体的基因蓝图。这种方法依赖于从复杂的人类微生物群体中特定地分离单个细胞或特定细胞群体，并对它们的基因组或其一部分进行测序，是对单个培养生物和全球群落元基因组学全基因组测序的补充。

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.