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.
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. ? ? ?
|Gole, Jeff; Gore, Athurva; Richards, Andrew et al. (2013) Massively parallel polymerase cloning and genome sequencing of single cells using nanoliter microwells. Nat Biotechnol 31:1126-32|
|Chen, Chun H; Cho, Sung H; Chiang, Hsin-I et al. (2011) Specific sorting of single bacterial cells with microfabricated fluorescence-activated cell sorting and tyramide signal amplification fluorescence in situ hybridization. Anal Chem 83:7269-75|
|Cho, Sung Hwan; Chen, Chun Hao; Tsai, Frank S et al. (2010) Mammalian Cell Sorting using ?FACS. Conf Lasers Electro Optics 2010:CTuD1|
|Cho, Sung Hwan; Chen, Chun H; Tsai, Frank S et al. (2010) Human mammalian cell sorting using a highly integrated micro-fabricated fluorescence-activated cell sorter (microFACS). Lab Chip 10:1567-73|
|Cho, Sung Hwan; Godin, Jessica; Lo, Yu-Hwa (2009) Optofluidic Waveguides in Teflon AF-Coated PDMS Microfluidic Channels. IEEE Photonics Technol Lett 21:1057-1059|
|Chen, Chun H; Cho, Sung Hwan; Tsai, Frank et al. (2009) Microfluidic cell sorter with integrated piezoelectric actuator. Biomed Microdevices 11:1223-31|
|Cho, Sung Hwan; Chen, Chun H; Tsai, Frank S et al. (2009) Micro-fabricated fluorescence-activated cell sorter. Conf Proc IEEE Eng Med Biol Soc 2009:1075-8|