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Absolute quantification of prokaryotes in the microbiome by 16S rRNA qPCR or ddPCR

Nature Protocols (2025)Cite this article

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Abstract

Measurements of prokaryotic absolute abundance can provide important insights into human gut microbiome biology and correct misinterpretations of relative abundance data. Despite the existence of several relatively well-established methods for making these measurements, most microbiome studies do not report absolute abundance. To enable researchers equipped with standard molecular biology capabilities to incorporate absolute quantification into their microbiome studies, we present a detailed, step-by-step protocol for rigorous and reproducible quantification of prokaryotic concentration in stool samples. We include methods for measuring stool sample moisture content, quantifying the concentration of the 16S rRNA prokaryotic marker gene by qPCR or digital droplet PCR (ddPCR) and analyzing the resulting data. We also highlight and provide strategies to overcome common pitfalls of the quantification method, such as 16S rRNA gene contamination. The final output of this approach is 16S rRNA copies per wet or dry gram of stool. In cases where samples have matched metagenomic sequencing information, data can be converted into absolute concentration of prokaryotes and taxon-specific absolute concentrations. To enable researchers to choose the appropriate method for their specific applications, we also compare and contrast our qPCR and ddPCR methods. In 4 days, ~80 samples can be taken from frozen stool to absolute concentration by using qPCR or ddPCR without the need for resequencing. Overall, this protocol provides a sensitive and straightforward way to measure the absolute concentration of prokaryotes in human gut microbiome samples stored with or without preservative.

Key points

  • This protocol quantifies prokaryotic concentration in stool samples by measuring 16S rRNA gene concentration with qPCR or ddPCR and correcting for stool sample moisture content.

  • Absolute prokaryotic quantification can provide further insights into microbiome biology and correct misinterpretations of the relative abundance data most commonly reported in microbiome studies.

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Fig. 1: Overview of the protocol to quantify 16S rRNA gene concentration in human stool samples.
Fig. 2: DNA extraction and stool sample moisture content measurement.
Fig. 3: 16S rRNA qPCR detailed schematic.
Fig. 4: 16S rRNA ddPCR detailed schematic.
Fig. 5: 16S rRNA qPCR and ddPCR comparison.

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Data availability

Source data for figures are available at https://github.com/bhattlab/absolute-abundance-16s.

Code availability

Data analysis scripts and notebooks are available at https://github.com/bhattlab/absolute-abundance-16s.

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Acknowledgements

We thank all members of the Bhatt Laboratory for discussions and experimental advice; K. Langenfeld for guidance on the ddPCR assay; A. Boehm for access to the ddPCR workflow equipment; D. Solow-Cordero and S. Sim for assistance in using the Stanford High-Throughput Bioscience Center and Stanford Functional Genomics Facility, which is supported by NIH Shared Instrumentation Grants S10RR019513, S10RR026338, S10OD025004 and S10OD026899 and by an anonymous donation; L. Nichols for guidance on flow cytometry microbial counting methods; and J. Axel for feedback on the manuscript, input on the figures and testing of the scripts. A.S.B. was supported by National Institutes of Health R01 AI148623 and R01 AI143757, a Distinguished Investigator Award from the Paul Allen Foundation and a Convergence grant from the Stand Up 2 Cancer Foundation. The Bhatt Laboratory is also supported by The Phil & Penny Knight Initiative for Brain Resilience at the Wu Tsai Neurosciences Institute, Stanford University. B.D. was supported by the Stanford Medical Scholars Fellowship Program, Stanford Berg Scholars Program and a Physician Scientist Institutional Award (PSIA) from the Burroughs Wellcome Fund. M.D. was supported by NIH Cellular and Molecular Biology Training Program Training Grant T32GM007276. D.G.M. was supported by the Stanford Gerald J. Lieberman Fellowship and the NIH Fogarty Global Health Equity Scholars Program (NIH FIC D43TW010540). Figures 1–4 were created with BioRender.com.

Author information

Author notes

  1. These authors contributed equally: Boryana Doyle, Gabriella Z. M. Reynolds, Mai Dvorak.

Authors and Affiliations

  1. School of Medicine, Stanford University, Stanford, CA, USA

    Boryana Doyle

  2. Department of Medicine (Hematology, Blood and Marrow Transplantation), Stanford University, Stanford, CA, USA

    Gabriella Z. M. Reynolds, Dylan G. Maghini, Aravind Natarajan & Ami S. Bhatt

  3. Department of Biology, Stanford University, Stanford, CA, USA

    Mai Dvorak

  4. Sydney Brenner Institute for Molecular Bioscience, University of the Witwatersrand, Johannesburg, South Africa

    Dylan G. Maghini

  5. Department of Genetics, Stanford University, Stanford, CA, USA

    Ami S. Bhatt

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Contributions

B.D., G.Z.M.R., M.D., A.N. and D.G.M. refined the approach for the qPCR and ddPCR assays. A.S.B., B.D., G.Z.M.R. and M.D. conceptualized the qPCR versus ddPCR comparison experiment. B.D., G.Z.M.R. and M.D. performed extraction, qPCR and ddPCR. B.D., G.Z.M.R. and M.D. carried out analysis and generated figures. B.D., G.Z.M.R., M.D. and A.S.B. wrote the manuscript with input from A.N. and D.G.M. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Ami S. Bhatt.

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Nature Protocols thanks the anonymous reviewers for their contribution to the peer review of this work.

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Key reference

Maghini, D. G. et al. Nat. Biotechnol. 42, 328–338 (2024): https://doi.org/10.1038/s41587-023-01754-3

Supplementary information

Supplementary Information

Supplementary Methods 1–4, Supplementary Figures 1–4

Supplementary Tables 1–3

Supplementary Tables 1–3

Supplementary Video 1

Video showing how to biopsy punch samples

Supplementary Video 2

Video showing how to vortex a ddPCR reaction plate before droplet generation

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Doyle, B., Reynolds, G.Z.M., Dvorak, M. et al. Absolute quantification of prokaryotes in the microbiome by 16S rRNA qPCR or ddPCR. Nat Protoc (2025). https://doi.org/10.1038/s41596-025-01165-5

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