Effects of 16S rRNA hypervariable region selection on respiratory microbiome profiling in healthy adults

Selection of the 16S rRNA gene target region can substantially influence inferred respiratory microbiome profiles, particularly in low-biomass samples. In this study, we compared full-length V1-V9 (Pacific Biosciences) and V3-V4 (Illumina) amplicon sequencing using exhaled breath condensate collected from 50 healthy adults. Samples that yielded successful amplification for both approaches were included in paired, within-subject comparisons of sequencing depth, alpha diversity, and taxonomic composition. Sequencing of the V3-V4 region yielded significantly higher read counts and greater alpha diversity (richness and Shannon index) than full-length V1-V9 sequencing (all p < 0.001). Both approaches consistently identified a shared set of dominant respiratory-associated genera, including Acinetobacter, Streptococcus, Psychrobacter, Gemella, Neisseria, and Granulicatella. However, relative abundance estimates differed by target region, with Acinetobacter enriched in V1-V9 profiles, whereas Streptococcus and Neisseria were more abundant in V3-V4 data. In contrast, several genera showed comparable abundances across regions. These findings indicate that while overall community membership is broadly conserved, quantitative diversity metrics and genus-level abundance estimates are strongly influenced by primer and target-region selection. Overall, these results provide methodological guidance for interpreting respiratory microbiome data derived from exhaled breath condensate and highlight the importance of aligning sequencing strategies with specific study objectives.