An Integrated lab-on-chip system for genome sequencing of single microbial cells

描述（由申请人提供）：人体内大约栖息着 100 万亿微生物。这些微生物与宿主之间的密切相互作用对人体的生理产生深远的影响。这些微生物中的大多数尚未完全表征，主要是由于在实验室条件下培养它们存在困难。在这里，我们建议开发一种有效且可扩展的方法来从单个微生物细胞中获取基因组序列。这将消除获得纯实验室培养物的需要，从而使我们能够系统地表征人体不同部位微生物群落的基因组结构。拟议项目包含三个主要部分。 1. 开发将微流体与微型光学元件集成的低成本一次性细胞分选设备。这样的芯片实验室细胞分选仪将能够从含有数百到数千种不同物种的样本中识别和分离单个微生物细胞，这些样本通常受到宿主和其他来源的游离 DNA 的污染。 2. 开发基于微孔的聚合酶克隆装置，用于同时并行扩增数百至数千个单个微生物细胞的基因组。使用这样的设备，我们将从单细胞中制备足够量的DNA用于全基因组鸟枪法测序，这是从单细胞获得基因组序列的关键步骤。 3. 开发用于以单细胞分辨率剖析人类微生物组基因组组成的集成管道。我们将通过从小鼠远端肠道中不同相对丰度水平的大约 35 个单个微生物细胞中分离和测序基因组来测试该管道。该方法将为研究界提供一种新工具来识别未知的微生物物种，研究其代谢功能并更好地了解各种生理和疾病条件下的宿主-微生物相互作用。公共卫生相关性：微生物在人体内的分布和活动对宿主的健康有着深远的影响。在这个拟议的项目中，我们将开发一种有效且可扩展的方法，从复杂微生物群落的单细胞中获取基因组序列。在详细研究它们的代谢功能和与宿主的相互作用之前，表征这些微生物的基因组组成和结构是必要的步骤。

DESCRIPTION (provided by applicant): There are approximately 100 trillion microorganisms inhabited in a human body. The intimate interactions between these microorganisms and the host have a profound impact of the physiology of the human body. The majority of these microorganisms have yet to be fully characterized, mainly due to the difficulties in growing them in laboratory conditions. Here we propose the development of an efficient and scalable method to obtain genome sequences from single microbial cells. This will eliminate the need to obtain pure laboratory culture, thus allow us to systematically characterize the genome structure of microbial communities that resides in different parts of the human body. The proposed project contains three major components. 1. Development of low-cost and disposable cell sorting devices that integrate microfluidics with micro- scale optical components. Such a lab-on-chip cell sorter will be able to identify and isolate single microbial cells from samples that contain hundreds to thousands of different species and often are contaminated with free DNAs from the host and other sources. 2. Development of a micro-well based polymerase cloning device for simultaneous amplification of genomes from hundreds to thousands of single microbial cells in parallel. Using such a device, we will prepare sufficient amount of DNAs from single cells for whole genome shotgun sequencing, a critical step to obtain genome sequences from single cells. 3. Development of an integrate pipeline for dissecting the genome composition of human microbiome at the single cell resolution. We will test this pipeline by isolating and sequencing genome from approximately 35 single microbial cells at different levels of relative abundance from the mouse distal intestine. The propose method will provide the research community a new tool to identify unknown microbial species, to study their metabolic functions and to better understand the host-microbe interactions under various physiological and disease conditions. PUBLIC HEALTH RELEVANCE: The distribution and activities of microorganisms in the human body have a profound impact on the health of the host. In this proposed project we will develop an efficient and scalable method to obtain genome sequences from single cells in complex microbial communities. Characterizing the genomic composition and structures of these microorganisms is a necessary step before their metabolic functions and the interactions with the host can be study in great detailed.