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Related Experiment Video

Updated: Mar 6, 2026

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing
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Critical Issues in Mycobiota Analysis.

Bettina Halwachs1, Nandhitha Madhusudhan2, Robert Krause3

  • 1Institute of Pathology, Medical University of GrazGraz, Austria; Theodor Escherich Laboratory for Medical Microbiome Research, Medical University of GrazGraz, Austria; BioTechMed-Graz, Interuniversity CooperationGraz, Austria.

Frontiers in Microbiology
|March 7, 2017
PubMed
Summary
This summary is machine-generated.

Analyzing fungal communities (mycobiota) is crucial for health. Standard bioinformatics tools, often designed for bacteria, struggle with fungal internal transcribed spacer (ITS) sequencing data, leading to inaccurate results.

Keywords:
16S rRNA geneDNA isolationOTU pickingformalin-fixed paraffin-embedded tissue (FFPE)internal transcribed spacer (ITS)microbiotamultiple sequence alignment (MSA)mycobiota

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Area of Science:

  • Microbiology
  • Bioinformatics
  • Genomics

Background:

  • Fungi are integral to the human microbiome, influencing health and disease.
  • Culture-independent methods have advanced mycobiota research, but analysis tools are often bacterial-centric.
  • Existing tools, primarily developed for bacterial 16S rRNA gene sequencing, are suboptimal for fungal internal transcribed spacer (ITS) marker gene analysis.

Purpose of the Study:

  • To provide a comprehensive overview of fungal amplicon sequencing analysis.
  • To highlight bioinformatics challenges, particularly in operational taxonomic unit (OTU) picking for mycobiota studies.
  • To evaluate the performance of standard analysis pipelines for fungal ITS data.

Main Methods:

  • Review of fungal amplicon analysis workflow from DNA extraction to bioinformatics.
  • In silico analysis using an ITS1 mock community.
  • Comparison of standard bioinformatics pipelines with a closed-reference OTU picking approach.

Main Results:

  • Standard bioinformatics pipelines, using default settings, produce inaccurate fungal community profiles.
  • Operational taxonomic unit (OTU) picking is a critical step where standard pipelines falter.
  • A closed-reference approach for OTU picking significantly improves the accuracy of fungal community representation.

Conclusions:

  • Current standard bioinformatics pipelines are inadequate for accurate fungal ITS amplicon analysis.
  • Optimized OTU picking strategies, such as closed-reference, are essential for reliable mycobiota studies.
  • Recommendations are provided for improved ITS-based mycobiota analysis using available methods.