There are many challenges to the study of the human enteric microbiome, among them solving practical issues such as obtaining undistorted and representative samples and determining how advanced technologies for discovery of uncharacterized, seemingly uncultivable, and poorly represented microorganisms can be applied to small sample sizes. However, studies to date have also failed to recognize that colonic lavage used to prepare the colon for standard colonoscopy significantly dilutes and distorts the enteric microbiome. Moreover, our knowledge of the human enteric microbiome is heavily based on analyses of stool and luminal samples which may not be sufficiently representative of the more residential and geographically-specific communities of mucosal- associated bacteria. In this regard, novel and underrepresented species that have special conditional or communal properties that facilitate their close proximity to the host are likely to be missed. These organisms are likely to have direct bearing on human health and disease. Regional differences in their composition and community organization must also be factored in when studying the human enteric microbiome. In this proposal, these issues will be taken into consideration in developing new and improved non-cultivation-based technologies that will ultimately facilitate genomic sequencing and metagenomic analysis of substantial numbers of previously uncharacterized members of the human enteric microbiome. We propose to obtain region-specific samples of mucosal associated microbes in their natural state within the unprepped human colon. We will then develop and refine two non-cultivation- based approaches aimed at obtaining high grade, composite DNA of microbial communities or enriched/purified samples of underrepresented, unclassified microbial species. The first involves laser capture microdissection of mucosal-associated bacteria from different regions of the human colon, which will be developed primarily for generating high quality DNA for metagenomic analyses. The second approach involves fluorescence in situ hybridization (FISH) using 16S rDNA and metagenomically-determined unique riboprobes coupled with fluorescence-activated cell sorting (FACS). Yield, enrichment, and purity will be optimized to discover and isolate novel, unclassified, and rare microbes from the human colonic microbiome for whole genome sequencing by high throughput sequencing centers. We believe these studies will produce non-cultivation-based technologies that will advance genomic sequencing and metagenomic analysis of substantial numbers of previously uncharacterized members of the human enteric microbiome.
This application is aimed at developing new and improved non-cultivation-based technologies that will ultimately facilitate genomic sequencing and metagenomic analysis of substantial numbers of previously uncharacterized members of the human enteric microbiome. This will be achieved through development of laser capture microdissection and FISH-FACS approaches on samples derived from intact, mucosal-associated microbial communities of the human enteric microbiome. ? ? ? ?
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