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Scientific reports2025; 15(1); 17037; doi: 10.1038/s41598-025-00969-5

Comparison of RNA- and DNA-based 16S amplicon sequencing to find the optimal approach for the analysis of the uterine microbiome.

Abstract: Studies in humans and large animals indicate a relationship between the uterine microbiome composition and endometrial receptivity. Despite many studies have been performed, the analysis of the uterine microbiome remains challenging due to the very low microbial biomass. Studies in other biological systems showed that RNA-based microbiome analysis complements DNA-based results and provides information about active bacteria in a sample. Thus, the aim of this study was to establish a highly sensitive and specific 16S rRNA gene V3-V4 amplicon PCR from equine uterine cytobrush samples and to compare DNA- and RNA-based 16S rRNA microbiome analysis. An optimized 16S rRNA gene V3-V4 amplicon PCR protocol from equine uterine cytobrush samples was developed, which was able to detect less than 38 bacterial genome copies using a bacterial DNA community standard. For the RNA-based amplicon generation protocol starting from cDNA, at least a 10-fold higher sensitivity was estimated compared to DNA-based approach. The comparison of using RNA and DNA isolated from the same uterine cytobrush samples as input for 16S V3-V4 amplicon sequencing revealed a much higher number of amplicon sequence variants as well as taxonomic units for the RNA-based approach. This resulted in significant differences in alpha (Simpson, Chao1) and beta diversity between RNA- and DNA-based analysis. Differential abundance analysis revealed significant differences between DNA and RNA samples at all taxonomic levels. Despite these differences, the overall microbiome composition was similar between the paired DNA and RNA samples. Many differences were probably found due to the higher sensitivity of the RNA-based approach. Furthermore, the DNA-based analysis is biased by the rRNA gene copy numbers (1-21), and the RNA-based analysis by the number of ribosomes per cell, which was reflected in the differences in the microbiome composition between the approaches. In addition, the results suggested that the DNA-based analysis is detecting cell-free bacterial DNA and/or DNA of dead bacteria that could be present in the samples. Altogether, the obtained results indicate advantages of a combined DNA- and RNA-based microbiome analysis, offering complementary and valuable information in the context of fertility-related studies of the uterine microbiome.
© 2025. The Author(s).
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Publication Date: 2025-05-16 PubMed ID: 40379732PubMed Central: PMC12084623DOI: 10.1038/s41598-025-00969-5Google Scholar: Lookup
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  • Journal Article
  • Comparative Study

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Research Overview

  • This study aimed to develop and compare RNA- and DNA-based 16S rRNA gene sequencing methods to analyze the uterine microbiome in horses, focusing on determining which approach offers greater sensitivity and insights into active bacterial populations.
  • The research highlights that RNA-based analyses detect more bacterial variants and may be more sensitive in identifying active bacteria than DNA-based analyses, which can detect both live and dead bacterial DNA.

Background and Motivation

  • The uterine microbiome affects endometrial receptivity and fertility in both humans and large animals.
  • Studying the uterine microbiome is challenging due to its very low microbial biomass, making sensitive detection methods necessary.
  • Previous research in other biological systems showed that RNA-based microbiome analyses provide complementary information by focusing on metabolically active bacteria, while DNA-based methods capture total bacterial presence, alive or dead.
  • This study aims to establish a sensitive protocol specifically for equine uterine samples and to compare RNA- versus DNA-based 16S rRNA gene sequencing of the uterine microbiome.

Methodology

  • Sample Collection: Equine uterine samples were collected using cytobrushes to obtain cells and associated bacteria.
  • PCR Optimization: Developed a highly sensitive PCR protocol targeting the V3-V4 region of the 16S rRNA gene optimized for low bacterial biomass samples.
  • Sensitivity Assessment: Used bacterial DNA community standards to quantify the limit of detection, achieving detection of fewer than 38 genome copies for the DNA-based method.
  • RNA-Based Approach: Converted RNA extracted from the samples into cDNA to perform PCR, estimating at least a 10-fold increased sensitivity compared to the DNA-based method.
  • Sequencing and Analysis: Performed 16S rRNA gene amplicon sequencing on paired RNA and DNA samples from the same uterine cytobrushes to directly compare diversity and composition results.

Key Findings

  • Higher Sensitivity of RNA-based Method: The RNA-derived cDNA approach detected a substantially higher number of amplicon sequence variants (ASVs) and taxonomic units.
  • Diversity Differences: Significant differences in alpha diversity metrics (Simpson, Chao1) and beta diversity were observed between RNA- and DNA-based analyses, indicating distinct microbial community profiles.
  • Microbiome Composition: Despite differences, overall bacterial community composition was broadly similar between paired RNA and DNA samples.
  • Taxonomic Differences: Differential abundance analysis showed significant variation at every taxonomic level between DNA and RNA samples.
  • Potential Biases in Methods: DNA-based results may be influenced by variable rRNA gene copy numbers (1-21 copies per genome) and may detect DNA from dead or cell-free bacteria.
  • RNA-based analysis: Reflects bacterial activity based on ribosome numbers within living cells, providing additional insight into active microbiota.

Interpretation and Implications

  • The RNA-based approach’s higher sensitivity likely accounts for detection of a greater diversity and more active members of the uterine microbiome.
  • The DNA-based method offers detection of total bacterial DNA, including dead organisms and free DNA, which may complicate interpretation regarding active bacterial populations.
  • Combining RNA- and DNA-based analyses captures complementary aspects of the uterine microbiome, encompassing both presence and activity of microbial communities.
  • This combined analysis is particularly valuable for fertility studies, where understanding metabolically active bacteria could lead to better insights into uterine health and reproductive success.
  • The optimized protocols developed in this study provide a methodological framework for sensitive and specific microbiome analysis in samples with low bacterial biomass.

Conclusions

  • This study establishes that RNA-based 16S rRNA sequencing is more sensitive and reveals a broader range of active bacteria in the uterine environment compared to DNA-based methods.
  • Significant differences in detected microbial diversity highlight methodological biases inherent in RNA versus DNA approaches.
  • Integration of RNA- and DNA-derived sequencing data yields complementary insights, suggesting a combined approach is optimal for comprehensive uterine microbiome characterization.
  • These findings provide important technical guidance and biological insights for future research on the role of the uterine microbiome in reproductive health.

Cite This Article

APA
Dyroff AI, López-Valiñas Á, Magalhaes HB, Podico G, Canisso IF, Almiñana C, Bauersachs S. (2025). Comparison of RNA- and DNA-based 16S amplicon sequencing to find the optimal approach for the analysis of the uterine microbiome. Sci Rep, 15(1), 17037. https://doi.org/10.1038/s41598-025-00969-5

Publication

Scientific reports
ISSN: 2045-2322
NlmUniqueID: 101563288
Country: England
Language: English
Volume: 15
Issue: 1
Pages: 17037
PII: 17037

Researcher Affiliations

Dyroff, Antonia I
  • Institute of Veterinary Anatomy, Vetsuisse Faculty Zurich, University of Zurich, Lindau (ZH), Switzerland. antoniaisabelle.dyroff@uzh.ch.
López-Valiñas, Álvaro
  • Institute of Veterinary Anatomy, Vetsuisse Faculty Zurich, University of Zurich, Lindau (ZH), Switzerland.
Magalhaes, Humberto B
  • College of Veterinary Medicine, University of Illinois Urbana-Champaign, Urbana, IL, USA.
Podico, Giorgia
  • College of Veterinary Medicine, University of Illinois Urbana-Champaign, Urbana, IL, USA.
Canisso, Igor F
  • College of Veterinary Medicine, University of Illinois Urbana-Champaign, Urbana, IL, USA.
Almiñana, Carmen
  • Institute of Veterinary Anatomy, Vetsuisse Faculty Zurich, University of Zurich, Lindau (ZH), Switzerland.
  • Department of Reproductive Endocrinology, University Hospital Zurich, Zurich, Switzerland.
Bauersachs, Stefan
  • Institute of Veterinary Anatomy, Vetsuisse Faculty Zurich, University of Zurich, Lindau (ZH), Switzerland. stefan.bauersachs@uzh.ch.

MeSH Terms

  • Female
  • Animals
  • RNA, Ribosomal, 16S / genetics
  • Microbiota / genetics
  • Uterus / microbiology
  • DNA, Bacterial / genetics
  • Horses
  • Bacteria / genetics
  • Bacteria / classification
  • Sequence Analysis, DNA / methods
  • RNA, Bacterial / genetics

Grant Funding

  • 200534 / Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung

Conflict of Interest Statement

Declarations. Competing interests: The authors declare no competing interests. Ethics approval and consent to participate: The samples were collected from mares owned by the University of Illinois Urbana-Champaign or from client-owned mares presented at the Veterinary Hospital (IACUC ethical approvals 21237 and 21238, respectively).

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Citations

This article has been cited 1 times.
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