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

• Published in: Applied microbiology and biotechnology Vol. 88; no. 6; pp. 1333 - 1342
• Main Authors: Wang, Yunwei, Antonopoulos, Dionysios A, Zhu, Xiaorong, Harrell, Laura, Hanan, Ira, Alverdy, John C, Meyer, Folker, Musch, Mark W, Young, Vincent B, Chang, Eugene B
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Berlin/Heidelberg : Springer-Verlag 01.12.2010; Springer-Verlag; Springer; Springer Nature B.V
• Subjects: Biodiversity; Biological and medical sciences; Biomedical and Life Sciences; Biopsy; Biotechnology; Carbohydrates; Cluster Analysis; Colon; Colon - microbiology; Communities; Community structure; Deoxyribonucleic acid; DNA; DNA sequencing; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; DNA, Ribosomal - chemistry; DNA, Ribosomal - genetics; Ethanol; Feasibility; Feasibility studies; feces; Fundamental and applied biological sciences. Psychology; Gastroenterology; Gene sequencing; Genes; Genomics; High-Throughput Nucleotide Sequencing; Host-microbe interactions; Human; Human Experimentation; Humans; Intestinal Mucosa - microbiology; Laser capture microdissection; Lasers; Life Sciences; Medical imaging; Metabolism; Metagenome; Metagenomics; Microbial activity; microbial communities; Microbial Genetics and Genomics; Microbiology; Microbiomes; Microdissection - methods; Microorganisms; Molecular Sequence Data; Mucosa; Mucosa-associated microbes; Multiple displacement amplification; Nucleic Acid Amplification Techniques; Nucleic acids; Nucleotide sequence; nucleotide sequences; Phylogeny; Proteomics; Pyrosequencing; Regional; RNA, Ribosomal, 16S - genetics; Sequence Analysis, DNA; Sequencing; Structure-function relationships; Studies; Transcriptomics; United States > US; Metagenomics; Host-microbe interactions; Laser capture microdissection; Multiple displacement amplification; Pyrosequencing; Mucosa-associated microbes; Human; Interaction; Mucosa; Amplification; Laser; Colon; Microorganism; Microbial community
• ISSN: 0175-7598; 1432-0614; 1432-0614
• DOI: 10.1007/s00253-010-2921-8

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.