Sample Preparation Using Pressure for Microbiome Studies and Clinical Diagnostics

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.