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Updated: Jun 22, 2025

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing
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Population sequencing for diversity and transmission analyses.

Talima Pearson1, Tara Furstenau1,2, Colin Wood1

  • 1Pathogen &Microbiome Institute, Northern Arizona University, Flagstaff, Arizona, United States of America.

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|July 1, 2024
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Population sequencing directly from mixed samples, combined with minimal colony sequencing, reveals pathogen genomic diversity without haplotype reconstruction. This method efficiently tracks disease spread and identifies transmission reservoirs.

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Area of Science:

  • Microbiology and Genomics
  • Population Genetics
  • Epidemiology

Background:

  • Genomic diversity in pathogen populations drives evolution, adaptation, and immune evasion.
  • Understanding population-level diversity is crucial for epidemiological tracking and transmission pathway analysis.
  • Traditional methods of culturing and sequencing numerous colonies are time-consuming and costly.

Purpose of the Study:

  • To present a novel method for characterizing pathogen population genomic diversity using population sequencing and minimal colony sequencing.
  • To demonstrate the utility of this method for phylogenetic analysis without requiring haplotype reconstruction.
  • To apply the method to study population structures and transmission dynamics of *Burkholderia pseudomallei* and *Staphylococcus aureus*.

Main Methods:

  • Developed and applied population sequencing directly from mixed clinical samples (sputum, body sites).
  • Combined population sequencing with sequencing of a small number of isolated colonies.
  • Analyzed genomic diversity and phylogenetic patterns in *Burkholderia pseudomallei* and *Staphylococcus aureus* populations.

Main Results:

  • Successfully captured and characterized phylogenetic diversity in mixed bacterial populations.
  • Discovered highly structured *Burkholderia pseudomallei* populations within sputum, suggesting sputum as a proxy for lung colonization.
  • Revealed transmission directionality and identified reservoirs for *Staphylococcus aureus* across different hosts and body sites.

Conclusions:

  • Population sequencing offers an efficient alternative to traditional methods for assessing pathogen genomic diversity.
  • Sputum sampling can non-invasively reveal pathogen population structure within the lungs.
  • This approach provides high-resolution insights into disease spread, transmission dynamics, and reservoir identification.