Comparison of Oral Collection Methods for 16S rRNA Gene and Shotgun Metagenomic Sequencing

We investigated how different sample collection methods affect oral microbiome studies by comparing unpreserved saliva, saliva preserved in glycerol, and mouthwash samples. We assessed their stability at room temperature and their intraindividual stability over six months. Samples were collected from 20 healthy participants at two time points six months apart; saliva samples were split, with half preserved in glycerol. Some samples were frozen immediately, while others were stored at room temperature for a week. DNA was extracted using PowerSoil Pro, followed by 16S rRNA gene and shotgun metagenomic sequencing. Intraclass correlation coefficients (ICCs) from taxonomic and functional tables were used to assess variability, and sample size requirements were calculated based on intraindividual stability. Saliva in glycerol more closely resembled unpreserved saliva than mouthwash, as reflected by higher median ICCs at genus (0.88 vs 0.60), species (0.92 vs 0.64), and gene levels (0.84 vs 0.36; all P<0.01). Room temperature storage had a greater effect on saliva in glycerol (median genus-level ICC=0.65) than mouthwash. No significant differences were observed at the gene level. Intraindividual stability over six months was moderate. To detect an odds ratio of 1.5 using one sample per individual, estimated sample sizes ranged from 665 for common species to 219,547 for rare species. Oral microbiome stability depends on the collection method; mouthwash offers better room temperature stability and may be more suitable where immediate freezing is not possible. For epidemiological studies, consistently using one collection method and longitudinal sampling can improve reproducibility and the ability to detect associations with health outcomes.]]>