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Pyrosequencing for Microbial Identification and Characterization
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A quantitative sequencing method using synthetic internal standards including functional and phylogenetic marker

Kazuyoshi Koike1, Ryo Honda2, Masataka Aoki3

  • 1Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa, Japan.

Environmental Microbiology Reports
|July 19, 2023
PubMed
Summary

Synthetic internal standard genes (ISGs) were developed for functional markers in amplicon sequencing. This method accurately quantifies microbial genes, including 16S rRNA, pmoA, and amoA, in environmental samples.

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Environmental Science

Background:

  • Synthetic internal standard genes (ISGs) are typically used for phylogenetic markers in amplicon sequencing.
  • Their application to functional markers, crucial for understanding microbial processes, has been limited.

Purpose of the Study:

  • To develop and validate ISGs for quantifying functional genes (pmoA, amoA) alongside the 16S rRNA gene.
  • To assess the accuracy of ISG-spiking for absolute quantification of microbial populations in environmental samples.

Main Methods:

  • Development of ISGs for 16S rRNA, pmoA, and amoA genes.
  • Spiking ISGs into mock and environmental samples.
  • Performing amplicon sequencing and quantitative real-time PCR (qPCR).
  • Analyzing sequencing data for absolute gene quantification.

Main Results:

  • ISG-based quantification closely matched qPCR for 16S rRNA and pmoA genes in mock communities.
  • A slight underestimation was observed for the amoA gene, potentially due to primer coverage.
  • The method was successfully applied to environmental sludge samples, revealing abundant Methylocystis populations.

Conclusions:

  • ISG spiking is a viable method for evaluating sequencing data processing and accurately quantifying functional gene markers.
  • This approach enhances the reliability of microbial community analysis, particularly for functional gene abundance.