Single Cell Genomics of Human Microbial Flora

描述（由申请人提供）：这份更新申请提议继续我们的工作，开发用于全基因组扩增的多重位移扩增（MDA）反应。MDA可用于从单个细胞中获得扩增的DNA，用于测序。这项研究将继续关注居住在人体中的微生物。新方法将演示扩增从人类临床标本（包括皮肤，喉咙和粪便样本）中获得的未培养细菌的单细胞DNA。为了减少扩增偏倚，将继续对丙二醛反应的酶学进行基础研究。微菌落技术也将用于通过提供来自多个细胞而不是单个细胞的DNA模板来减少扩增偏差。将继续研究流式细胞术和显微操作分离单个细胞的更好方法。荧光原位杂交（FISH）探针将用于分离感兴趣的特定微生物。这些方法将被引入我们的项目，为NIH人类微生物组计划提供扩增的基因组DNA测序。还将更加强调医学上的重要问题，包括分析定殖于人类的正常细菌群落和涉及医院获得性感染的病原体。我们将展示单细胞方法来研究导致医院获得性感染的胃肠道病原体艰难梭菌（与加州大学圣地亚哥医学中心合作）。还将介绍对未培养病毒和真核生物进行测序的方法。将与Illumina公司和加州大学圣地亚哥分校的Pavel Pevzner合作开发改进的信息学方法，以组装来自MDA反应的序列。

DESCRIPTION (provided by applicant): This renewal application proposes to continue our work to develop the multiple displacement amplification (MDA) reaction which is used for whole genome amplification. MDA can be used to obtain amplified DNA from single cells for use in sequencing. The study will continue to focus on microbes inhabiting the human body. New methods will be demonstrated for amplifying DNA from single cells of uncultured bacteria obtained from human clinical specimens including skin, throat and fecal samples. Basic research will continue into the enzymology of the MDA reaction in order to reduce amplification bias. Microcolony technology will also be used to reduce amplification bias by providing DNA template from multiple cells rather than a single cell. Research will continue into better methods to isolate individual cell by flow cytometry and micromanipulation. Fluorescent in situ hybridization (FISH) probes will be tested for isolating specific microbes of interest. These methods will be introduced into our program to supply amplified genomic DNA for sequencing by the NIH Human Microbiome Project. There will also be increased emphasis on medically important questions including analysis of the normal bacterial community colonizing humans and pathogens involved in hospital acquired infections. We will demonstrate single cell methods for investigating a GI tract pathogen, C. difficile, that causes hospital acquired infections (in collaboration with the University of California at San Diego Medical Center). Methods to sequence uncultivated viruses and eukaryotes will also be introduced. Improved informatic methods to assemble sequences derived from MDA reactions will be developed in collaboration with Illumina Inc. and Pavel Pevzner, UCSD. PUBLIC HEALTH RELEVANCE: This renewal application continues our work to develop the multiple displacement amplification (MDA) reaction which is used to amplify DNA. MDA can be used to obtain amplified DNA from single cells for use in sequencing. More than one billion copies of DNA are produced starting with the single DNA copy contained in a bacterial cell. This study will continue to focus on microbes inhabiting the human body. New methods will be demonstrated for amplifying DNA from single cells of uncultured bacteria obtained from human clinical specimens including skin, throat and fecal samples. For the many bacteria that cannot be grown in culture, MDA will make it possible to obtain enough DNA for use in sequencing their genomes.