Genomics, Transcriptomics, Proteomics

Applied Microbiology and Biotechnology

, Volume 88, Issue 6, pp 1333-1342

Laser capture microdissection and metagenomic analysis of intact mucosa-associated microbial communities of human colon

  • Yunwei WangAffiliated withDepartment of Medicine, Knapp Center for Biomedical Discovery, University of Chicago
  • , Dionysios A. AntonopoulosAffiliated withDepartment of Medicine, Knapp Center for Biomedical Discovery, University of ChicagoInstitute for Genomics and Systems Biology, Argonne National Laboratory
  • , Xiaorong ZhuAffiliated withDepartment of Medicine, Knapp Center for Biomedical Discovery, University of Chicago
  • , Laura HarrellAffiliated withDepartment of Medicine, Knapp Center for Biomedical Discovery, University of Chicago
  • , Ira HananAffiliated withDepartment of Medicine, Knapp Center for Biomedical Discovery, University of Chicago
  • , John C. AlverdyAffiliated withDepartment of Surgery, University of Chicago
  • , Folker MeyerAffiliated withInstitute for Genomics and Systems Biology, Argonne National Laboratory
  • , Mark W. MuschAffiliated withDepartment of Medicine, Knapp Center for Biomedical Discovery, University of Chicago
  • , Vincent B. YoungAffiliated withDivision of Infectious Diseases, University of Michigan
    • , Eugene B. ChangAffiliated withDepartment of Medicine, Knapp Center for Biomedical Discovery, University of Chicago Email author 

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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.

Keywords

Laser capture microdissection Metagenomics Mucosa-associated microbes Multiple displacement amplification Pyrosequencing Host-microbe interactions