Stability of the Fecal and Oral Microbiome over 2 Years at −80°C for Multiple Collection Methods

In prospective cohorts, biological samples are generally stored over long periods before an adequate number of cases have accrued. We investigated the impact of sample storage at -80°C for 2 years on the stability of the V4 region of the 16S rRNA gene across seven different collection methods (i.e., no additive, 95% ethanol, RNAlater stabilization solution, fecal occult blood test cards, and fecal immunochemical test tubes for feces; OMNIgene ORAL tubes and Scope mouthwash for saliva) among 51 healthy volunteers.Intraclass correlation coefficients (ICC) were calculated for the relative abundance of the top three phyla, the 20 most abundant genera, three alpha-diversity metrics, and the first principal coordinates of three beta-diversity matrices.The subject variability was much higher than the variability introduced by the sample collection type, and storage time. For fecal samples, microbial stability over 2 years was high across collection methods (range, ICCs = 0.70-0.99), except for the samples collected with no additive (range, ICCs = 0.23-0.83). For oral samples, most microbiome diversity measures were stable over time with ICCs above 0.74; however, ICCs for the samples collected with Scope mouthwash were lower for two alpha-diversity measures, Faith's phylogenetic diversity (0.23) and the observed number of operational taxonomic units (0.23).Fecal and oral samples in most used collection methods are stable for microbiome analyses after 2 years at -80°C, except for fecal samples with no additive.This study provides evidence that samples stored for an extended period from prospective studies are useful for microbiome analyses.