Targeted genomic characterization of uncultured bacteria from the human microbiot

描述（由申请人提供）：人类微生物组项目的主要目标是鉴定与人体相关的所有生物体（人类微生物群），并确定其中大多数（如果不是全部）的基因组序列。检测到的人类微生物群的多样性达到数千种物种和菌株，其中绝大多数尚未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，用于在单细胞水平上对人类微生物群的任何未培养成分进行靶向基因组表征，并允许对可能具有一些培养分离株的选定生物群进行群体遗传学研究。我们的策略利用高系统发育分辨率的小亚基核糖体RNA（SSU rRNA）提供区分微生物的核糖体类型。我们计划标记和分离代表未培养的微生物谱系的单细胞以及来自复杂微生物群样本的特定微生物类型的细胞群体，并将其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个体培养生物体的测序与整体群落鸟枪测序（宏基因组学）之间的差距，整体群落鸟枪测序通常不提供足够的深度和分辨率来全面测序微生物组。初步的可行性研究表明，我们的方法可以应用于任何微生物财团，是不依赖于目标生物的丰度。在此基础上，该提案的重点是确定用于来自人类微生物群的微生物的靶向单细胞和群体基因组学的最佳实验设计和改进的技术程序。具体目标是：1.目标1。从肠道微生物群样品中分离单细胞和目标未培养微生物菌群类型的群体。我们将使用荧光原位杂交（FISH）结合流式细胞术，以获得单细胞和群体的靶向细胞类型，未培养或与少数培养的代表2。目标二。来自代表未培养的肠道微生物群的单细胞的基因组的扩增和测序。我们将使用多重置换扩增法扩增靶细胞的基因组，并对DNA进行测序以获得基因组组装草图。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：未培养和培养的微生物菌群类型的目标群体的泛基因组学表征。我们将分离代表未培养生物体的特定细菌亚类的群体以及代表在培养中具有代表性的种/属的细胞群体，并对一个或几个基因组进行测序。我们将扩增和测序细胞群体基因组DNA，以获得复合基因组/泛基因组。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获得居住在人体内的任何生物体的遗传蓝图。这种方法依赖于从复杂的人类微生物聚生体中特异性分离单细胞或特定细胞群并对其基因组或其一部分进行测序，补充了来自个体培养生物体和全球社区宏基因组学的全基因组测序。

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.