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Updated: May 15, 2025
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Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing
Published on: October 15, 2019
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Advancements in analytical methods for studying the human gut microbiome
Gijsbert J Jansen1, Gerard P Schouten1, Marit Wiersma1
1NL-Lab, Biotrack, Leeuwarden, Friesland, 8912 AP Netherlands.
Journal of Biological Methods
|April 9, 2025
Summary
Studying the human gut microbiome requires careful selection of analytical techniques. Understanding the strengths and limitations of methods like PCR, NGS, and FISH is crucial for accurate microbiome research.
Area of Science:
- Microbiology
- Genomics
- Bioinformatics
Background:
- The human gut microbiome is a complex microbial ecosystem vital for health.
- Alterations in gut microbiome composition are associated with various diseases.
Purpose of the Study:
- To review and compare common analytical techniques used in gut microbiome research.
- To highlight the strengths and limitations of each method for accurate microbiome analysis.
Main Methods:
- Polymerase Chain Reaction (PCR): Sensitive but DNA extraction dependent.
- Next-Generation Sequencing (NGS): Powerful but costly and data-intensive; accuracy relies on DNA extraction quality.
- Culture Methods: Useful but biased and time-consuming.
- Fluorescence In Situ Hybridization (FISH): Visualizes specific microbes in situ, but traditionally limited by human interpretation and sensitivity.
Main Results:
- Each technique presents unique advantages and disadvantages.
- DNA extraction quality significantly impacts PCR and NGS accuracy.
- FISH offers unique in situ visualization but faced throughput limitations.
Conclusions:
- Choosing the appropriate analytical technique is critical for reliable gut microbiome research.
- Awareness of method-specific limitations is essential for interpreting results and drawing robust conclusions.
