ORIGINAL RESEARCH article
Front. Cell. Infect. Microbiol.
Sec. Oral Microbes and Host
Comparative analysis of full-length 16s ribosomal RNA gene sequencing in human oropharyngeal swabs using primer sets with different degrees of degeneracy
Provisionally accepted
Christian Waechter1,2,3*
Janna Nele Wilkens2Leon Fehse4
Dominik Heider4Kiarash Sassani5Georgios Chatzis2Sebastian Weyand6Sabine Pankuweit2Ulrich Luesebrink2
Muhidien Soufi2Jochen Pöling3
Thomas Braun3
Felix Ausbuettel2
Volker Ruppert2*- 1Department of Cardiology III, University Hospital Münster, Münster, Germany
- 2Philipps-Universitat Marburg, Marburg, Germany
- 3Max-Planck-Institut fur Herz- und Lungenforschung W G Kerckhoff-Institute, Bad Nauheim, Germany
- 4Universitat Munster, Münster, Germany
- 5Universitat Greifswald, Greifswald, Germany
- 6Universitat Ulm, Ulm, Germany
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Abstract Background: Full-length 16S rRNA gene sequencing using nanopore technology has become increasingly relevant for profiling complex microbial communities, including the human oral microbiome. Primer selection plays a critical role in amplification bias and taxonomic resolution, yet remains insufficiently investigated for oropharyngeal samples. Methods: We conducted a comparative analysis of two primer sets with differing degrees of degeneracy – Oxford Nanopores (ONT) standard 27F primer (27F-I) and a more degenerate variant (27F-II) – for full-length 16S rRNA gene sequencing of 80 human oropharyngeal swab samples using ONTs MinION Mk1C. Alpha diversity and taxonomic profiles were statistically compared between primer sets and benchmarked against a large-scale salivary microbiome dataset (n=1,989) from healthy individuals. Results: Primer choice significantly impacted microbial community composition and diversity. The more degenerate primer set 27F-II yielded significantly higher alpha diversity (Shannon index: 2.684 vs. 1.850; p < 0.001) and detected a broader range of taxa across all phyla. The taxonomic profiles generated with 27F-II strongly correlated with the reference dataset (Pearson's r = 0.86, p < 0.0001), whereas profiles generated with 27F-I showed weak correlation (r = 0.49, p = 0.06). 27F-I overrepresented Proteobacteria and underrepresented key genera such as Prevotella, Faecalibacterium, and Porphyromonas. Conclusion: Our findings demonstrate that primer degeneracy has a substantial effect on taxonomic resolution and biodiversity estimates in oropharyngeal 16S rRNA gene sequencing. The more degenerate 27F-II primer set seams to more faithfully captures the complexity of the human oropharyngeal microbiome and aligns more closely with population-level reference data. These results underscore the importance of careful primer selection and support the adoption of degenerate primers as a methodological standard in nanopore-based oral microbiome research.
Keywords: 16S rRNA, oral microbiome, human oropharyngeal microbiome, next-generation sequencing (NGS), nanopore sequencing, Oxford Nanopore Technologies (ONT), MinION Mk1C
Received: 11 Jul 2025; Accepted: 27 Oct 2025.
Copyright: © 2025 Waechter, Wilkens, Fehse, Heider, Sassani, Chatzis, Weyand, Pankuweit, Luesebrink, Soufi, Pöling, Braun, Ausbuettel and Ruppert. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence:
Christian Waechter, christian.waechter@staff.uni-marburg.de
Volker Ruppert, ruppert@med.uni-marburg.de
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