Single Cell Genomics of Human Microbial Flora

描述（由申请人提供）：本更新申请建议继续我们的工作，以开发用于全基因组扩增的多重置换扩增（MDA）反应。MDA可用于从单细胞获得扩增的DNA以用于测序。这项研究将继续关注居住在人体内的微生物。新的方法将被证明用于扩增来自人类临床标本（包括皮肤、喉咙和粪便样本）的未培养细菌单细胞的DNA。基础研究将继续进入MDA反应的酶学，以减少扩增偏倚。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模板来降低扩增偏倚。将继续研究通过流式细胞术和显微操作分离单个细胞的更好方法。将检测荧光原位杂交（FISH）探针，以分离感兴趣的特定微生物。这些方法将被引入到我们的计划中，以提供扩增的基因组DNA供NIH人类微生物组计划测序。还将更加重视医学上的重要问题，包括对人类正常细菌群落和医院获得性感染中涉及的病原体的分析。我们将演示用于调查胃肠道病原体C.艰难梭菌，其导致医院获得性感染（与加州大学圣地亚哥医学中心合作）。还将介绍未培养的病毒和真核生物的测序方法。将与Illumina Inc.合作开发改进的信息学方法，以组装来自MDA反应的序列。帕维尔·佩夫兹纳，加州大学圣地亚哥分校

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.