Long-term stability of oral samples in the freezer for microbiome analyses
Background: 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 of buccal cells and saliva over five years to provide insight into the stability of oral samples in the freezer for future microbiome analyses.Methods: Healthy participants provided buccal cell 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 and the V4 region of the 16S rRNA gene sequenced and additional aliquots were then similarly extracted and sequenced after being stored for approximately one month, three months, six months, one year, and five years after collection. Intraclass correlation coefficients were calculated for four alpha-diversity metrics, the first two principal coordinates of four beta-diversity matrices, and the 14 most abundant genera.Results: The alpha diversity ICCs for both sample types remained stable over five years. For beta diversity there was a slight downward trend in the ICCs for some PCoA vectors from some of the matrices but nevertheless the beta diversity the ICCs were good or excellent. The ICCs for the relative abundances of the genera during the five years of freezer storage were stable with only a couple of exceptions.Conclusion: Both buccal cell and saliva samples are relatively stable for microbiome analysis after five years when stored at -80C