Processing Method Affects Oral and Gut Microbiome Composition

Abstract Objectives Conducting research on the microbiome has become prevalent, particularly in the area of precision nutrition as nutritional strategies targeting the microbiome are increasingly explored. However, differences in sample processing methods for generating microbial genetic sequencing data could influence observed microbial composition. The objective of this study was to process human oral and gut samples using several common methods to examine the influence of each on microbiota composition. Methods We collected fecal, saliva, and dental swab samples from 10 individuals. Fecal samples were 1) fresh, 2) lyophilized (freeze-dried), 3) frozen then ground in liquid nitrogen, or 4) stored at room temperature (RT) for 72 hours, frozen, then ground in liquid nitrogen. Saliva and dental swabs were 1) fresh (saliva), 2) subjected to the Human Microbiome Project (HMP) initial processing protocol (saliva), or 3) frozen whole upon collection and split into different fractions after thawing: supernatant, pellet, or mixed supernatant and pellet (both saliva and dental). We performed 16S rRNA gene sequencing, used QIIME2 to identify amplicon sequence variants (ASVs) and calculate diversity metrics, and used MaAsLin2 to evaluate the effect of treatment types on relative abundances of microbes. Results For fecal samples, we found significant differences in the relative abundances of 51 ASVs between fresh and lyophilized, four ASVs between fresh and liquid nitrogen post-72 hour at RT, and two ASVs between fresh and liquid nitrogen treated samples. There were differences among both saliva and dental swab fractions. For saliva, the relative abundances of 15 ASVs differed significantly between both mixed and pellet fractions and mixed and supernatant fractions. For dental swabs, the relative abundances of 19 ASVs differed significantly between mixed and pellet fractions and 10 between mixed and supernatant fractions. For all reported significant results, q < 0.05. Conclusions Relative abundances are significantly affected by processing methods for both gut and oral microbiome samples. Therefore it is crucial to exercise caution when comparing microbial composition results across studies that use different processing methods. Funding Sources Research reported in this abstract was supported by a NIH NIDDK T32 Award.