Sample Preparation Using Pressure for Microbiome Studies and Clinical Diagnostics

描述（由申请人提供）：高度平行的DNA测序仪和高通量质谱仪的引入正在推动微生物学进入一个新时代，将其重点从研究培养物中单个生物体的特性扩展到理解微生物群落中物种之间复杂的相互关系。人类微生物组计划（Human Microbiome Project，HMP）是人类基因组计划的逻辑概念和实验延伸，旨在了解人类微生物区系，其遗传和生理多样性（微生物组）以及影响其组成微生物的分布和进化的因素。HMP的第一个任务是获得微生物组有机体的全基因组序列。随后，这些生物体的转录组学、蛋白质组学和代谢组学数据将在各种生理和病理条件下进行生化解释研究。在这些研究和未来诊断应用中有用的技术中，样品制备是数据采集和解释中最重要的瓶颈和错误来源之一。有一种假设是，样品制备的第一步破坏了微生物组的每个成员，并允许几乎定量分离感兴趣的生物分子。然而，这种完整和有效的样品制备可能是一项具有挑战性的任务，特别是对于粪便样品，粪便样品是人体中发现的各种微生物的栖息地和具有压倒性化学复杂性的样品基质。在目前的SBIR第一阶段项目中，我们建议基于我们专有的压力循环技术样品制备系统（PCT SPS）开发专门的样品制备方法。我们的目的是进行可行性研究的提取和定量回收的DNA从难以裂解的微生物粪便样品。另一个目的是研究基于微生物亚群的机械和理化性质逐步破坏和分离DNA的可行性。该任务旨在简化复杂微生物混合物的分析，从而使下游基因组分析变得更有效。我们的长期目标是建立一个适用于广泛微生物组研究和未来临床诊断的样品制备平台。公共卫生相关性：该研究旨在开发一种专门的压力循环技术平台，用于从粪便等复杂样品中提取总DNA，以促进NIH人类微生物组项目等研究，该项目旨在阐明生活在人体内和人体上的微生物的性质。这些项目旨在通过研究这些微生物如何促进正常生理和疾病来最终改善公共健康。这项技术的发展与我们对微生物组的理解以及药物发现和未来的临床诊断高度相关。

DESCRIPTION (provided by applicant): The introduction of highly parallel DNA sequencers and high throughput mass spectrometers is propelling microbiology into a new era, extending its focus from studying the properties of single organisms in culture to the understanding of intricate inter-relationship of species in microbial communities. The Human Microbiome Project (HMP), a logical conceptual and experimental extension of the Human Genome Project, aims to understand the human microflora, its genetic and physiological diversity (the Microbiome) and the factors that influence the distribution and evolution of its constituent microorganisms. The first task in HMP is the acquisition of the whole genome sequences of the microbiomic organisms. Later, the transcriptomic, proteomic and metabolomic data of these organisms will be studied with biochemical interpretations under various physiological and pathological conditions. Among technologies useful in these studies and for the future diagnostic applications, sample preparation is one of the most significant bottlenecks and sources of errors in data acquisition and interpretation. There is an assumption that the first step of sample preparation disrupts every member of the microbiome and permits nearly quantitative isolation of the biomolecules of interest. However, such complete and efficient sample preparation can be a challenging task, particularly for fecal samples, which are the habitat of the diverse variety of microorganisms found in the human body and a sample matrix of overwhelming chemical complexity. In the current SBIR Phase I project, we propose to develop specialized sample preparation methods based on our proprietary Pressure Cycling Technology Sample Preparation System (PCT SPS). We aim to conduct feasibility studies on the extraction and quantitative recovery of DNA from difficult-to-lyse microorganisms in fecal samples. Another objective is to study the feasibility of a stepwise disruption and isolation of DNA from microbial subgroups based on their mechanical and physiochemical properties. This task aims to simplify the analysis of complex microorganism mixtures, so that downstream genomic analysis becomes more efficient. Our longer term objective is to establish a sample preparation platform applicable in broad microbiome research and future clinical diagnostics. PUBLIC HEALTH RELEVANCE: The proposed study aims to develop a specialized Pressure Cycling Technology platform for extraction of total DNA from complex samples such as feces, in order to facilitate research such as the NIH Human Microbiome Project, which aims to elucidate the nature of the microbial organisms that live in and on the human body. Such projects aim to ultimately improve public health by examining how these microbes contribute to normal physiology and disease. Development of this technology is highly relevant to advancing our understanding of the microbiome, as well as for drug discovery and future clinical diagnostics.