Cross-platform comparison of oral microbiome profiles under a unified reference database

The primary goal of this study is to systematically characterize the inherent biases between Illumina short-read and Oxford Nanopore Technologies (ONT) long-read sequencing platforms for oral microbiome analysis. While previous comparisons have reported varying degrees of concordance, interpretation has often been confounded by the use of different reference databases and analysis pipelines. In this project, we reanalyzed paired human oral samples (saliva and interdental plaque) under a unified framework using the expanded Human Oral Microbiome Database (eHOMD) to control for database-driven variation. We specifically aimed to quantify platform-specific discrepancies in relative abundance across taxonomic ranks and to evaluate the fundamental capability of each platform to achieve species-level taxonomic assignment. By elucidating persistent systematic biases, such as the significant overestimation of Actinomycetota by Illumina and the superior species-level resolution of ONT, this study provides essential guidance for researchers in selecting the appropriate platform and critically interpreting 16S rRNA gene amplicon data for oral microbiome research.