Laser capture microdissection and metagenomic analysis of intact mucosa-associated microbial communities of human colon |
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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 |
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Institution: | (1) Department of Medicine, Knapp Center for Biomedical Discovery, University of Chicago, Rm 9031, 900 E. 57th Street, Chicago, IL 60637, USA;(2) Department of Surgery, University of Chicago, 5841 S. Maryland Ave, Chicago, IL 60637, USA;(3) Institute for Genomics and Systems Biology, Argonne National Laboratory, 9700 S. Cass Avenue, Argonne, IL 60439, USA;(4) Division of Infectious Diseases, University of Michigan, 4618D Med Sci II SPC 5623, 1150 W. Medical Center Dr, Ann Arbor, MI 48109, USA |
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Abstract: | 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. |
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