Long-term stability at -80°C of oral wash and saliva samples for microbiome analyses

Large-scale prospective biological studies necessitate the storage of oral samples for numerous years to accrue adequate sample sizes. However, there is minimal research on the impact of long-term storage of oral samples on the oral microbiome. We investigated the freezer stability over 5 years of the oral microbiome measured from oral wash and saliva samples to provide insight for future microbiome analyses of stored oral samples. Healthy participants provided oral wash and saliva samples using Scope mouthwash and the OMNIgene ORAL collection device, respectively. DNA was extracted from an aliquot of each sample type at baseline, the V4 region of the 16S rRNA gene sequenced, and additional aliquots were then similarly extracted and sequenced after being stored for approximately 1 month, 12 months, and 5 years after collection. Intraclass correlation coefficients (ICC) and 95% confidence intervals (CI) were calculated for 4 alpha-diversity metrics, the first 2 principal coordinates of four beta-diversity matrices, and the 14 most abundant genera. The alpha diversity and beta diversity ICCs for both sample types remained stable over 5 years. For example, the 5-year Shannon index ICCs were 0.94 (95% CI: 0.88, 0.98) and 0.90 (95% CI: 0.72, 0.95) for oral wash and saliva samples, respectively. The ICCs for the relative abundances of the examined genera during the 5 years of freezer storage were also generally stable. Both oral wash and saliva samples were relatively stable for diversity metrics and relative abundance after 5 years when stored at -80°C.IMPORTANCELarge, prospective studies will likely need to store biospecimens for many years in the freezer prior to DNA extraction and sequencing for analyses considering the association between the microbiome and specific health conditions. In this study, we demonstrated that oral wash using Scope mouthwash and saliva specimens in the OMNIgene ORAL kit have generally stable microbiome communities for up to 5 years at -80°C.