Authors: Yunwei Wang Dionysios A Antonopoulos Xiaorong Zhu Laura Harrell Ira Hanan John C Alverdy Folker Meyer Mark W Musch Vincent B Young Eugene B Chang
Publish Date: 2010/10/08
Volume: 88, Issue: 6, Pages: 1333-1342
Abstract
Metagenomic analysis of colonic mucosaassociated 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 mucosaassociated 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 mucosaassociated 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 GSFLX DNA sequencer Metagenomic analysis of approximately 67 million base pairs of DNA sequences from two samples revealed that the metabolic functional profiles in mucosaassociated 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 mucosaassociated microbial communities at small spatial scalesWe 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 5R01GM06234411 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 researchPhylogenetic relationships among the OTUs detected in LCM DNA samples before and after phi29 amplification The representative 61 clone numbers are listed in the tree from right colon and 72 clone numbers are listed in the tree from left colon Red branches were clones obtained from 16S rRNA gene library built up from unamplified DNA and blue branches were clones from 16S rRNA gene library built up from phi29amplified DNA Sequences were aligned with RDP and are given the name at the Genus level Bootstrap values are based on 100 replications GIF 23 kb
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