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Modern Molecular Taxonomy01:29

Modern Molecular Taxonomy

Advancements in molecular biology have revolutionized the identification and characterization of bacteria, with multiple methods leveraging DNA sequencing for enhanced precision. As sequencing technologies improve and costs decline, these approaches are increasingly used in clinical, environmental, and evolutionary studies.Multilocus Sequence Typing (MLST) examines several housekeeping genes, essential chromosomal genes encoding cellular functions, to distinguish strains. Approximately...
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Guided Protocol for Fecal Microbial Characterization by 16S rRNA-Amplicon Sequencing
08:05

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Published on: March 19, 2018

Microarray analysis and barcoded pyrosequencing provide consistent microbial profiles depending on the source of

Bartholomeus van den Bogert1, Willem M de Vos, Erwin G Zoetendal

  • 1Laboratory of Microbiology, Wageningen University, Dreijenplein 10, 6703 HB Wageningen, Netherlands.

Applied and Environmental Microbiology
|January 25, 2011
PubMed
Summary

High-throughput sequencing and microarray analysis provide comparable insights into intestinal microbiota composition. Primer selection impacts bacterial detection, but overall conclusions on microbial communities remain consistent across methods.

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Pyrosequencing for Microbial Identification and Characterization
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Published on: August 22, 2013

Area of Science:

  • Microbiology
  • Genomics
  • Bioinformatics

Background:

  • Characterizing the intestinal microbiota is crucial for understanding human health.
  • High-throughput 16S rRNA gene-based technologies are essential for large-scale microbiota analysis.

Purpose of the Study:

  • To compare barcoded pyrosequencing and phylogenetic microarray analysis (HITChip) for intestinal microbiota profiling.
  • To assess the impact of different forward primers on microbial profiling results.
  • To investigate discrepancies between the two techniques.

Main Methods:

  • Analysis of bacterial composition in fecal and small intestinal samples using pyrosequencing and HITChip.
  • PCR amplification with different forward primers for 16S rRNA gene sequencing.
  • Quantitative PCR and cloning/sequencing for validation.

Main Results:

  • Pyrosequencing yielded an average of 7,944 sequences per sample.
  • Primer choice affected Actinobacteria detection but not species richness or diversity estimates.
  • Microbial profiles from pyrosequencing and HITChip showed strong correlation for fecal and ileal samples, but less for ileostomy effluent.
  • Divergence in ileostomy effluent profiling was attributed to database limitations in HITChip probe design.

Conclusions:

  • High-throughput profiling of microbial communities yields equivalent biological conclusions regardless of technology or primer choice.
  • Primer selection can influence the detection of specific bacterial groups.
  • Database completeness is critical for accurate microbiota profiling, especially for less-studied sample types.