Species-by-Species Dissection of Microbiomes using Phage Display and Flow Sorting

描述（由申请人提供）：宏基因组学是近几年发展起来的一门新的科学学科。它既是一套研究技术，包括许多相关的途径和方法，也是一个研究领域。作为一个科学领域，宏基因组学试图解决四个层次的问题：1）在一个特定的复杂微生物组中存在什么微生物，如人类肠道？2)比例是多少？3)他们在做什么以及4）他们将如何对环境变化（例如饮食的变化）作出反应？目前，回答这些问题的方法是蛮力鸟枪测序和16S序列调查。然而，这些技术只能提示存在哪些种类的细菌，并且提供的信息往往偏向于最常见的物种。复杂微生物组中的大多数细菌目前无法以常规方式培养和测序。来自单细胞的全基因组扩增（WGA）已用于若干研究中。然而，在最好的情况下，只有60%的基因组可以被WGA从单个细胞中获得的DNA覆盖。研究表明，WGA的偏倚是随机的，可以通过增加相同基因组的更多拷贝来提高覆盖率。我们在这里建议改变宏基因组学的范式：而不是在没有任何独立信息的情况下批量提取所有DNA，我们建议开发能够逐个分析物种的工具。这将通过使用噬菌体展示来选择识别群体中的物种的抗体，然后使用这样选择的抗体通过流式细胞术表征物种的丰度，纯化它，并且如果必要的话耗尽物种的群体以重复该过程来进行。纯化的细菌将用作全基因组扩增、通过rRNA分析进行物种表征和测序（如有必要）的起始材料。本提案中开发的抗体将用于进行所示分析。在人类肠道微生物组分析的背景下开发的那些在细菌组成可能发挥病因学作用的临床研究的背景下也将非常有用。将E.大肠杆菌和其他几种细菌将在方法开发的第一阶段使用，人类肠道中的微生物群将在项目的后期部分进行分析。公共卫生相关性：人类与生活在其中的细菌合作存在，其中大多数是良性的。然而，最近的研究表明，微生物组的组成可能对个体的健康产生深远的影响。通过开发新的工具来分析人体微生物组，我们将提供额外的方法来研究和表征不同的细菌，并阐明它们在人类疾病中的作用。

DESCRIPTION (provided by applicant): Metagenomics is a new scientific discipline that has developed in the last several years. It is both a set of research techniques, comprising many related approaches and methods, and a research field. As a scientific field, metagenomics attempts to resolve four tiers of questions: 1) what micro-organisms are present in a particular complex microbiome, such as human gut? 2) in what proportions? 3) what they are doing? and 4) how will they react to environmental changes, such as a change in diet? Currently, the approach to answer these questions has been one of brute force shotgun sequencing and 16S sequence surveys. However these technologies can only hint as to which kinds of bacteria are present, and the information provided tends to be biased to the commonest species. The majority of bacteria in complex microbiomes cannot presently be cultured and sequenced in a conventional way. Whole genome amplification (WGA) from single cells has been used in several studies. However, in the best cases, only 60% of a genome can be covered with the DNA obtained with WGA from a single cell. Studies have showed that the bias from WGA is random and coverage can be improved by adding more copies of the same genome. We propose here to change the metagenomics paradigm: rather than extracting all DNA in bulk without any independent information on the species that comprise it, we propose to develop tools to be able to analyze species one by one. This will be carried out by using phage display to select antibodies that recognize species in the population, and then to use such selected antibodies to characterize the abundance of the species by flow cytometry, purify it, and if necessary deplete the population of the species in order to repeat the process. The purified bacteria will be used as starting material for whole genome amplification, species characterization by rRNA analysis, and sequencing, if necessary. The antibodies developed within this proposal will be used to carry out the analyses indicated. Those developed within the context of the analysis of the human gut microbiome will also be very useful within the context of clinical studies in which bacterial composition may play an etiological role. An artificial bacterial mixture of E. coli and several other bacterial species will be used at the first stage of method development, and the microbiota in human gut will be analyzed in the later portion of the project. PUBLIC HEALTH RELEVANCE: Humans exist in collaboration with the bacteria that live within them, most of which are benign. However, it has recently been shown that the composition of the microbiome can have profound effects on the health of individuals. By developing new tools to analyze human microbiomes, we will provide additional methods to study and characterize different bacteria and elucidate their role in human disease.