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

  • Genomics, Transcriptomics, Proteomics
  • Published:
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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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Acknowledgments

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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Authors and Affiliations

  1. Department of Medicine, Knapp Center for Biomedical Discovery, University of Chicago, Rm 9031, 900 E. 57th Street, Chicago, IL, 60637, USA

    Yunwei Wang, Dionysios A. Antonopoulos, Xiaorong Zhu, Laura Harrell, Ira Hanan, Mark W. Musch & Eugene B. Chang

  2. Department of Surgery, University of Chicago, 5841 S. Maryland Ave, Chicago, IL, 60637, USA

    John C. Alverdy

  3. Institute for Genomics and Systems Biology, Argonne National Laboratory, 9700 S. Cass Avenue, Argonne, IL, 60439, USA

    Dionysios A. Antonopoulos & Folker Meyer

  4. Division of Infectious Diseases, University of Michigan, 4618D Med Sci II SPC 5623, 1150 W. Medical Center Dr, Ann Arbor, MI, 48109, USA

    Vincent B. Young

Authors
  1. Yunwei Wang
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  2. Dionysios A. Antonopoulos
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  3. Xiaorong Zhu
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  4. Laura Harrell
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  5. Ira Hanan
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  6. John C. Alverdy
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  7. Folker Meyer
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  8. Mark W. Musch
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  9. Vincent B. Young
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  10. Eugene B. Chang
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Corresponding author

Correspondence to Eugene B. Chang.

Additional information

Yunwei Wang and Dionysios A. Antonopoulos have contributed equally to the paper.

Electronic supplementary material

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Supplementary Fig. 1

Minimal contamination of host eukaryotic DNA by LCM technique. Host epithelial cell mixed in mucus layer is distinct by its red staining and separated first before microdissecting the mucosa-associated microbes (×400). Arrow shows one epithelial cell in the mucus layer (PDF 2123 kb)

Supplementary Fig. 2

Reproducibility of T-RFLP method. To investigate the reproducibility of T-RFLP method in this study, 16S rRNA gene was amplified from the same DNA template using three independent PCR reactions and digested by MspI. Digested PCR products were dialysed and sequenced. Variations within three profiles were evaluated. Fragment size in base pairs is shown at the top and relative peak heights are shown as relative fluorescence. GeneScan-500 internal size standard and 5′ terminal restriction fragments are shown as red and blue peaks, respectively. The level of methodological variability was analyzed by calculating peak position and peak area from the triplicate profiles. The variation from triplicate PCR reactions from the same DNA sample ranged from 1.7% to 3.2% which implied a high reproducibility of this method. (PDF 226 kb)

Supplementary Fig. 3

Phylogenetic 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 un-amplified DNA and blue branches were clones from 16S rRNA gene library built up from phi29-amplified 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)

High-resolution image (TIFF 4217 kb)

Supplementary Fig. 4

Relative distribution of classified sequences related to carbohydrate metabolism in human colon samples. Note the inverse representation of the majority of the sub-classes in the two samples (PDF 57 kb)

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Wang, Y., Antonopoulos, D.A., Zhu, X. et al. Laser capture microdissection and metagenomic analysis of intact mucosa-associated microbial communities of human colon. Appl Microbiol Biotechnol 88, 1333–1342 (2010). https://doi.org/10.1007/s00253-010-2921-8

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  • DOI: https://doi.org/10.1007/s00253-010-2921-8

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