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Human colonic biota studied by ribosomal DNA sequence analysis

K H Wilson1, R B Blitchington

  • 1Infectious Diseases Section, VA Medical Center, Durham, NC 27705, USA. Wilso003@mc.duke.edu

Applied and Environmental Microbiology
|July 1, 1996
PubMed
Summary

Comparing culture methods with direct sequencing of human colonic biota revealed that minimizing PCR cycles preserves microbial diversity. This approach improves agreement between culturing bacteria and direct ribosomal DNA sequencing for accurate ecosystem representation.

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Genomics

Background:

  • The human colonic microbiota is a complex microbial ecosystem crucial for host defense.
  • Culture-based methods have been traditionally used to study its composition.
  • Direct sequencing methods offer an alternative for microbial ecosystem analysis.

Purpose of the Study:

  • To compare the effectiveness of established culture-based methods with direct amplification and sequencing of 16S rRNA genes for analyzing human colonic biota.
  • To evaluate the impact of different Polymerase Chain Reaction (PCR) cycle numbers on the representation of microbial diversity.

Main Methods:

  • A human fecal specimen was analyzed using both quantitative culture and direct amplification of 16S ribosomal DNA (rDNA) genes.
  • Partial sequencing of cloned 16S rRNA genes was performed on amplicons generated by 9 and 35 PCR cycles.

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  • Ribosomal DNA sequences were classified by comparing them to known phylogenetic sequences.

    • Main Results:

    • Quantitative culture recovered 58% of the microscopically counted bacteria.
    • Direct sequencing after 35 PCR cycles resulted in a distorted ecosystem representation with reduced diversity (13 sequences, 74% coverage).
    • Minimizing PCR cycles (9 cycles) preserved biodiversity (27 sequences, 59% coverage) and showed better agreement with culturing methods.

    Conclusions:

    • Minimizing PCR cycles during direct ribosomal DNA sequencing is crucial for preserving the biodiversity of the human colonic ecosystem.
    • Direct sequencing with optimized PCR conditions provides a more accurate representation of the colonic microbiota compared to high-cycle PCR methods.
    • This optimized direct sequencing approach aligns well with traditional culturing techniques for studying gut bacteria.