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

描述（由申请人提供）：大约有100万亿个微生物居住在人体内。这些微生物与宿主之间的密切相互作用对人体的生理机能有着深远的影响。这些微生物中的大多数尚未得到充分表征，主要是由于在实验室条件下培养它们很困难。在这里，我们提出了一种有效的和可扩展的方法来获得基因组序列从单个微生物细胞的发展。这将消除获得纯实验室培养的需要，从而使我们能够系统地表征驻留在人体不同部位的微生物群落的基因组结构。拟议的项目包括三个主要部分。1. 开发集微流体与微尺度光学元件于一体的低成本、一次性细胞分选装置。这种芯片上的实验室细胞分选器将能够从含有数百到数千种不同物种的样品中识别和分离单个微生物细胞，这些样品通常被来自宿主和其他来源的游离dna污染。2. 基于微孔的聚合酶克隆装置的开发，用于同时扩增数百至数千个单个微生物细胞的基因组。利用该装置，我们将从单细胞中制备足够数量的dna进行全基因组鸟枪测序，这是获得单细胞基因组序列的关键一步。3. 在单细胞分辨率上对人类微生物组的基因组组成进行解剖的整合管道的开发。我们将通过从小鼠远端肠中分离和测序大约35个不同相对丰度的单个微生物细胞来测试这一管道。该方法将为研究未知微生物种类、研究其代谢功能以及更好地了解各种生理和疾病条件下宿主-微生物相互作用提供新的工具。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。在这个项目中，我们将开发一种高效和可扩展的方法，从复杂微生物群落的单细胞中获得基因组序列。在详细研究这些微生物的代谢功能和与宿主的相互作用之前，确定这些微生物的基因组组成和结构是必要的一步。

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.