Comparative analysis of full-length 16s ribosomal RNA gene sequencing in human oropharyngeal swabs using primer sets with different degrees of degeneracy

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.