Laser capture microdissection and metagenomic analysis of intact mucosa-associated microbial communities of human colon
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Metagenomic analysis of colonic mucosa-associated microbes has been complicated by technical challenges that disrupt or alter community structure and function. In the present study, we determined the feasibility of laser capture microdissection (LCM) of intact regional human colonic mucosa-associated microbes followed by phi29 multiple displacement amplification (MDA) and massively parallel sequencing for metagenomic analysis. Samples were obtained from the healthy human subject without bowel preparation and frozen sections immediately prepared. Regional mucosa-associated microbes were successfully dissected using LCM with minimal contamination by host cells, their DNA extracted and subjected to phi29 MDA with a high fidelity, prior to shotgun sequencing using the GS-FLX DNA sequencer. Metagenomic analysis of approximately 67 million base pairs of DNA sequences from two samples revealed that the metabolic functional profiles in mucosa-associated microbes were as diverse as those reported in feces, specifically the representation of functional genes associated with carbohydrate, protein, and nucleic acid utilization. In summary, these studies demonstrate the feasibility of the approach to study the structure and metagenomic profiles of human intestinal mucosa-associated microbial communities at small spatial scales.
KeywordsLaser capture microdissection Metagenomics Mucosa-associated microbes Multiple displacement amplification Pyrosequencing Host-microbe interactions
We thank Kevin White and the Institute for Genomics and Systems Biology at the University of Chicago for their support and technical assistance. We would like to acknowledge the following grant support: Digestive Disease Research Core Center (P30 DK42086; EBC), R21HG004858 (EBC), R01 HG004906 (VY), UH3 UH3DK083993 (VY), The Goldgraber fellowship foundation (LL), R01 5R01GM062344-11 (JA), and Research Fellowship Award (YW) from the Crohn’s and Colitis Foundation of America. We would also like to thank the Gastrointestinal Research Foundation of Chicago and Peter and Carol Goldman for supporting the microbiome research.
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