Massively Parallel Single Cell Genomics of the Human Microbiome

描述（由申请人提供）：估计自然生活在人体内和人体表面的100万亿微生物的数量超过人体细胞的十倍。由NIH赞助的人类微生物组项目旨在更好地了解这些复杂的群落对人类生理，营养，免疫和发育的影响。这些细菌在健康和疾病中起着重要作用，但令人惊讶的是，由于无法培养和研究其中许多细菌，人们对它们知之甚少。研究人类微生物组的一种方法是分析人口中每个成员的基因和基因组。SBIR第一阶段资助提案“人类微生物组的大规模平行单细胞基因组学”旨在开发高通量方法，用于分离大量单个微生物细胞，扩增单个微生物细胞的基因组，并构建无克隆文库以进行大规模的序列分析。这些目标将通过开发简化单细胞基因组学的DNA扩增工具，显着提高现有无克隆文库构建方法的效率，并将这些方法与用于操纵单细胞及其基因组的新液滴平台整合来实现。一种新的热稳定噬菌体DNA聚合酶，在高温下快速有效地扩增DNA，克服了单细胞基因组学的许多技术挑战。一种将测序文库的构建提高两个数量级的新方法将简化和加速这些基因组的分析。一种新的基于液滴的微流控反应装置，精确控制单细胞的操作和筛选，提高单分子的扩增效率，并使每个实验能够并行处理1000万个液滴，有望加速许多生物分子方法的发展。该提案的长期目标是完善和整合这些工具和仪器，以便在易于处理的规模上研究人类微生物组。公共卫生相关性：SBIR第一阶段拨款提案“人类微生物组的大规模并行单细胞基因组学”旨在整合新的工具和技术来分析生活在人类体内和体表的数万亿微生物的基因和基因组。可以预测的结果包括新的健康诊断生物标志物，包括人类微生物群成员及其产生的化学信使的21世纪药典，基于人类微生物群产生的酶的工业应用，以及对人类营养需求的更深入了解。将特定基因或基因组与疾病状态相关联的能力也将与公共卫生相关。

DESCRIPTION (provided by applicant): The 100 trillion microorganisms that naturally live inside and on humans are estimated to outnumber human cells by a factor of ten. The Human Microbiome Project sponsored by the NIH seeks to better understand the effects these complex communities have on human physiology, nutrition, immunity and development. These bacteria play an important role in health and disease, but surprisingly little is known about them due to the inability to cultivate and study many of them. One way to study the human microbiome is to analyze the genes and genomes of each member of the population. The SBIR Phase I grant proposal "Massively Parallel Single Cell Genomics of the Human Microbiome" seeks to develop high throughput methods for isolating large numbers of individual microbial cells, amplifying the genomes of single microbial cells and constructing clone-free libraries for sequence analysis on a numerically large scale. These goals will be achieved by developing DNA amplification tools that simplify single-cell genomics, dramatically improving the efficiency of existing clone-free library construction methods, and integrating these methods with a new droplet based platform for manipulating single cells and their genomes. A new thermostable phage DNA polymerase that rapidly and efficiently amplifies DNA at high temperature overcomes numerous technical challenges for single cell genomics. A new method that improves the construction of sequencing libraries by two orders of magnitude will simplify and accelerate the analysis of these genomes. A new droplet based microfluidic reaction device that precisely controls the manipulation and screening of single cells, improves the amplification efficiency of single molecules and enables the parallel processing of 10 million droplets per experiment promises to accelerate the development of numerous biomolecular methods. The long term goal of the proposal is to refine and integrate these tools and instruments in order to study the human microbiome on a scale that is tractable. PUBLIC HEALTH RELEVANCE: The SBIR Phase I grant proposal "Massively Parallel Single Cell Genomics of the Human Microbiome" seeks to integrate new tools and technologies to analyze the genes and genomes of the trillions of microbes that live in and on humans. Outcomes that can be predicted include new diagnostic biomarkers of health, a twenty-first century pharmacopoeia that includes members of the human microbiota and the chemical messengers they produce, industrial applications based on enzymes that are produced by the human microbiota, and a deeper understanding of the nutritional requirements of humans. The ability to correlate particular genes or genomes with a disease state will also be relevant to public health.