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To develop high-throughput sequencing techniques as a useful method for transcription profile analysis through method development. Explore new or young technologies that may be useful as qualitative and/or quantitative techniques for analyzing mixed bacterial populations found in vivo and applying these new methods and techniques to evaluate the microecology of the host-bacteria system.
Approach: This project will develop methods that can elucidate the molecular metabolic details of complex, in vivo, mixed bacterial systems. It is of primary importance to develop methods that will enable the extraction of high quality RNA from these complex systems, the purification of subgroups of RNA (e.g., polyA, rRNA, mRNA, prokaryotic RNA), and the synthesis of double stranded cDNA from RNA. It is also important to pursue alternate means of analyzing RNA that may lead to more precise analyses in the future, such as adapting existing tools to analyze high throughput sequence data (i.e., 454) data. Other methods such as quantitative RT-PCR, microarrays, microbial sequencing, and proteomics will need to be used to compare and evaluate the results obtained using high throughput pyrosequencing.