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相关的所有生物体（人类微生物群），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群的多样性达到数千种和菌株，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任何未培养的人类微生物群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物的选定生物体群体进行群体遗传研究。我们的策略利用小亚基核糖体RNA （SSU rRNA）在区分微生物种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种型细胞群体，并将其DNA扩增到能够进行基因组测序的水平。这种方法将弥补有限数量的单个培养生物测序和整个群落霰弹枪测序（宏基因组学）之间的差距，后者通常不能提供足够的深度和分辨率来对微生物组进行全面测序。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联合体，而不依赖于目标生物的丰度。基于此，本提案的重点是确定最佳的实验设计和改进的技术程序，用于从人类微生物群中靶向单细胞和群体基因组学的微生物。具体目标是：1。目的1。从肠道菌群样本中分离单细胞和目标未培养的微生物类群。我们将使用荧光原位杂交（FISH）结合流式细胞术获得未培养或培养较少的目标种型的单细胞和群体2。目标2。代表未培养肠道菌群的单细胞基因组扩增和测序。我们将使用多重位移扩增扩增靶细胞的基因组，并对DNA进行测序，以获得初步的基因组组装。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。非培养和培养微生物种型目标群体的全基因组特征。我们将分离代表未培养生物的特定细菌种型群体，以及代表在培养中有代表并进行了一个或几个基因组测序的物种/属的细胞群体。我们将对细胞群体基因组DNA进行扩增和测序，获得复合基因组/泛基因组。公共卫生相关性：未培养微生物群的靶向基因组学将使有选择性地获得居住在人体中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群落中特异性分离单细胞或特定细胞群，并对其基因组或部分基因组进行测序，对单个培养生物的全基因组测序和全球群落宏基因组学进行补充。

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.