ArticleFrontiers in cellular and infection microbiology2025
Comparative analysis of full-length 16s ribosomal RNA gene sequencing in human oropharyngeal swabs using primer sets with different degrees of degeneracy.
Article in Frontiers in cellular and infection microbiology, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 2 papers.
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2 citing papers in PubMed.
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- Effect of sialidase inhibitors on a plaque community biofilm model.Access microbiology · 2026Article
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14 authors.
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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.
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