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

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.

Fig. 1

Procedure for the study of mucosa-associated microbial communities using metagenomic methods

Fig. 2

Laser capture microdissection (LCM) to harvest mucosa-associated microbes (×400). a By Alcian blue and nuclear fast red staining, the mucus layer (blue) is clearly different from mucosal epithelial cells (pink). b Mucosa-associated microbes are separated by LCM. c 16S rRNA gene PCR and T-RFLP analysis shows that representative microbes are collected by LCM from human colon mucosa. Richness (r) and evenness (e) were calculated based on the number and height of peaks in each profile

Fig. 3

Optimization of phi29 multiple displacement amplification (MDA) with complex DNA templates. a 16S rRNA gene PCR and T-RFLP analysis of fidelity of MDA with the different amount of DNA templates. High fidelity of MDA is observed in the reactions using more than 1 ng DNA as template. Richness (r) and evenness (e) were calculated to show the differences between samples b Dendrogram based on similarity analysis of T-RFLP profiles shows the relationships among samples. The scale bar shows the distance of similarity. c Optimization of Phi29 MDA time on laser capture microdissected DNA samples. Within 3-h amplification, microgram quantity DNA is harvested with minimum endogenous background amplification

Fig. 4

Overview of fidelity of phi29 MDA on laser capture micro-dissected DNA samples using 16S rRNA gene library cloning and sequencing. Bacterial composition is compared before and after phi29 MDA, based on the alignment with RDP. Operational taxonomic units and Shannon diversity were calculated based on the 16S rRNA gene sequences at the 97% cutoff value

Fig. 5

Distribution of classified metagenomic-derived sequences obtained from human colon samples. Genomic DNA was isolated from the mucosa of biopsy samples and subjected to phi29 polymerase multiple displacement amplification (MDA). Note the difference in representation of genes associated with DNA and protein metabolism between the samples obtained from the right colon relative to the left colon sample