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

描述(申请人提供)：元基因组学是近几年发展起来的一门新学科。它既是一套研究技术，包含了许多相关的途径和方法，又是一个研究领域。作为一个科学领域，元基因组学试图解决四个层次的问题：1)在特定的复杂微生物群中，如人的肠道中存在哪些微生物？2)按多大比例存在？3)它们在做什么？以及4)他们将如何应对环境变化，例如饮食的变化？目前，回答这些问题的方法之一是暴力猎枪测序和16S序列调查。然而，这些技术只能暗示存在哪些种类的细菌，提供的信息往往偏向最常见的物种。目前，复杂微生物群中的大多数细菌都不能以常规方式培养和测序。来自单个细胞的全基因组扩增(WGA)已被用于多项研究。然而，在最好的情况下，用WGA从单个细胞获得的DNA只能覆盖一个基因组的60%。研究表明，来自WGA的偏差是随机的，通过增加相同基因组的更多拷贝可以提高覆盖率。我们建议改变元基因组学范式：我们建议开发工具，能够逐个分析物种，而不是批量提取所有DNA，而不是在没有关于组成它的物种的任何独立信息的情况下提取所有DNA。这将通过使用噬菌体展示来选择识别种群中物种的抗体来实现，然后使用这些选择的抗体通过流式细胞术来表征物种的丰度，纯化它，如果必要的话，耗尽物种的种群以重复这一过程。纯化的细菌将被用作全基因组扩增的起始材料，通过rRNA分析鉴定物种，并在必要时进行测序。在这项提案中开发的抗体将用于进行所示的分析。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起病因学作用的临床研究的背景下也将非常有用。在方法开发的第一阶段将使用大肠杆菌和其他几种细菌的人工细菌混合物，并将在项目的后期分析人类肠道中的微生物区系。与公共卫生相关：人类与生活在其中的细菌合作生存，其中大多数是良性的。然而，最近的研究表明，微生物群的组成可以对个人的健康产生深远的影响。通过开发分析人类微生物群的新工具，我们将提供更多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.