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Absolute quantification of prokaryotes in the microbiome by 16S rRNA qPCR or ddPCR
Boryana Doyle1, Gabriella Z M Reynolds2, Mai Dvorak3
1School of Medicine, Stanford University, Stanford, CA, USA.
Nature Protocols
|May 19, 2025
Summary
This study provides a detailed protocol for measuring prokaryotic absolute abundance in gut microbiome samples using qPCR or digital droplet PCR (ddPCR). This method ensures accurate quantification, crucial for understanding microbiome biology and avoiding misinterpretations of relative abundance data.
Area of Science:
- Microbiology
- Molecular Biology
- Bioinformatics
Background:
- Accurate prokaryotic absolute abundance measurements are vital for human gut microbiome research.
- Most studies rely on relative abundance, which can lead to misinterpretations.
- Existing methods for absolute quantification are not widely adopted.
Purpose of the Study:
- To present a detailed, step-by-step protocol for reproducible absolute quantification of prokaryotes in stool samples.
- To enable researchers with standard molecular biology tools to perform absolute abundance measurements.
- To compare quantitative PCR (qPCR) and digital droplet PCR (ddPCR) methods for this application.
Main Methods:
- Measurement of stool sample moisture content.
- Quantification of 16S rRNA gene concentration using qPCR or ddPCR.
- Analysis of data, including strategies to overcome common pitfalls like contamination.
- Conversion of data to absolute prokaryote concentration per gram of stool.
Main Results:
- The protocol yields 16S rRNA copies per wet or dry gram of stool.
- Absolute prokaryote and taxon-specific concentrations can be derived with metagenomic data.
- The protocol allows processing of approximately 80 samples in 4 days without resequencing.
- Both qPCR and ddPCR methods are presented and compared.
Conclusions:
- This protocol offers a sensitive and straightforward method for measuring absolute prokaryote concentration in human gut microbiome samples.
- It facilitates more accurate interpretation of microbiome data by providing absolute abundance.
- The method is applicable to stool samples stored with or without preservative.
