Single Cell Genomics of Human Microbial Flora

描述(申请人提供)：一种名为多重置换扩增(MDA)的新方法使从单个细胞中对细菌基因组DNA进行测序成为可能。丙二醛被用来放大细胞中为数不多的DNA毫微克，用于测序、诊断和基因分型分析。丙二醛正迅速被用于环境细菌群落的调查，例如在海洋中，它可以被用来发现新的微生物。然而，将丙二醛用于研究生活在人体内的细菌的努力还很少，可以开发出强大的新研究策略来研究正常和致病的菌群。我们的目标是开发使用丙二醛从人类临床标本中扩增细菌基因组所需的方法。将开发用于组织、粪便样本、体液和其他标本类型的方法，分离单个细胞，裂解它们的DNA，并进行丙二醛全基因组扩增。初步研究表明，用流式细胞术高通量分离内耳粘膜中的流感嗜血杆菌。单细胞的分离也是通过显微操作进行的，在显微镜下使用玻璃毛细管，通过荧光DNA探针标记特定的细菌。芯片上的微流控丙二醛正在开发中，用于“护理点”诊断测试，这种测试可以在任何地方使用，而不需要实验室处理。激光捕获显微切割(LCM)正在作为一种替代细胞分离方法进行测试，用于受感染的组织，特别是在怀疑有细菌生物膜或细胞内感染的情况下。其他要研究的病原体包括来自中耳的金黄色葡萄球菌、肺炎链球菌和铜绿假单胞菌，以及来自伤口和受感染的医疗植入物的喉咙组织和猫。地面制动方法正在开发中，用于将组织和相关细菌的共聚焦显微镜成像与单细胞分离和基因组测序相结合。来自粪便样本的细菌将被用作圣路易斯华盛顿大学一个重大项目的一部分，该项目旨在识别人类肠道的微生物。莱姆病的病因伯氏疏螺旋体也已从扁虱的肠道中分离出来，DNA测序和基因分型证明了这一点。下一步计划从人类皮肤特有的莱姆病皮疹中分离出来，最终从被怀疑会导致神经症状的人脑组织中分离出来。在开发这些新应用程序的同时，我们在开发和优化MDA方法方面的工作仍在继续。对丙二醛反应的基本酶学和参与DNA扩增的化学途径进行了研究。在改进丙二醛的性能和完善利用Sanger测序法和454焦磷酸测序法的扩增DNA的方法和策略方面正在取得快速进展。总的目标是完成单细胞基因组DNA体外扩增方法的开发，并展示这项新技术在自然人类细菌群和传染病方面的研究应用。

DESCRIPTION (provided by applicant): A new method called Multiple Displacement Amplification (MDA) has made it possible to sequence bacterial genomic DNA from a single cell. MDA is used to amplify the few femtograms of DNA in the cell for use in sequencing and for diagnostic and genotyping assays. MDA is rapidly being adopted for investigation of environmental bacterial communities, such as in oceans, where it can be used to discover novel microbes. However, there has been little effort yet to use MDA for studying bacteria living in the human body where powerful new research strategies could be developed to study the normal and pathogenic flora. Our objective is to develop the methods needed for use of MDA in amplifying bacterial genomes from human clinical specimens. Methods will be developed for tissues, fecal samples, body fluids and other specimen types for isolating single cells, lysing them to release their DNA, and carrying out MDA whole genome amplification. Preliminary studies have demonstrated high throughput isolation of Haemophilus influenzae from inner ear mucosa by flow cytometry. Isolation of single cells is also being carried out by micromanipulation in which a glass capillary is used under the microscope and specific bacteria are targeted by labeling with fluorescent DNA probes. Microfluidic MDA on chips is being developed for use in "point of care" diagnostic tests that could be used at any location without lab processing required. Laser capture microdissection (LCM) is being tested as an alternative method of cell isolation for use with infected tissues, particularly where bacterial biofilms or intracellular infection are suspected. Other pathogens to be studied will be Staphylococcus aureus, Streptococcus pneumoniae, and Pseudomonas aeruginosa from middle ear, and throat tissues and puss from wounds and infected medical implants. Ground braking methods are in development for combining confocal microscopy imaging of tissues and associated bacteria with single cell isolation and genomic sequencing. Bacteria from fecal samples will be used as part of a major project at Washington University in St. Louis to identify the microbes of the human gut. Borrelia burgdorferi, the cause of Lyme disease, has also been isolated from tick gut and DNA sequencing and genotyping demonstrated. Isolation from the characteristic Lyme disease rash in human skin is planned next and ultimately from human brain tissue where it is suspected of causing neurological symptoms. In parallel with the development of these new applications, our work in developing and optimizing the MDA method continues. Research is proposed into the basic enzymology of the MDA reaction and the chemical pathways involved in DNA amplification. Rapid progress is being made to improve the performance of MDA and to perfect methods and strategies for use of amplified DNA by both the Sanger sequencing method and 454 pyrosequencing. The overall goal is to complete the development of methods for in vitro amplification of genomic DNA from singles cells and to demonstrate the research applications of this new technology to the natural human bacterial flora and to infectious disease.