Comparison of 16S rRNA gene amplicon and whole-genome shotgun metagenomic sequencing for subgingival oral microbiome profiling.

Background: Periodontitis is a chronic inflammatory disease driven by a dysbiotic subgingival microbiome. While 16S rRNA gene amplicon sequencing is widely used, whole-genome shotgun (WGS) metagenomics is increasingly applied for higher taxonomic and functional resolution. Objective: The aim of this study was to directly compare 16S rRNA gene amplicon (V1-V2) sequencing and WGS metagenomic sequencing using matched subgingival plaque samples from patients with periodontitis. Methods: Subgingival plaque samples from 28 patients with periodontitis were analyzed using both 16S rRNA gene amplicon (V1-V2) sequencing and WGS metagenomics. Taxonomic composition, microbial diversity, differential abundance and functional analysis were compared across platforms. Results: WGS generated markedly higher read counts than 16S rRNA gene amplicon but showed wide variability in non-human reads, whereas 16S rRNA gene amplicon yielded a consistent proportion of non-chimeric reads. High taxonomic overlap was observed at the phylum level but declined at higher taxonomic ranks. WGS preferentially detected taxa such as Conclusions: This comparative analysis demonstrates that 16S rRNA gene amplicon (V1-V2) sequencing and WGS both robustly capture core subgingival microbial signatures. While WGS provides higher species-level and functional resolution, the resolution was strongly constrained by microbial read depth in host-rich subgingival samples. These findings provide practical guidance for selecting appropriate sequencing strategies and optimizing sample preparation when designing WGS-based periodontal microbiome studies.