Systematic analysis of sampling regions and storage methods on fecal gut microbiome and metabolome profiles

The contribution of human gastrointestinal (GI) microbiota and metabolites to host health has recently become much clearer. However, many confounding factors can influence the accuracy of gut microbiome and metabolome studies resulting in inconsistencies in published results. In this study, we systematically investigated the effects of fecal sampling regions, and storage and retrieval conditions on gut microbiome and metabolite profiles from three healthy children. Our analysis indicated that compared to homogenized and snap frozen samples (standard control: SC), different sampling regions did not affect microbial community alpha diversity, while a total of 22 of 176 identified metabolites significantly varied across different sampling regions. In contrast, storage conditions significantly influenced the microbiome and metabolome. Short-term room temperature storage had a minimal effect on the microbiome and metabolome profiles. Sample storage in RNALater showed a significant level of variation in both microbiome and metabolome profiles, independent of the storage or retrieval conditions. We conclude that homogenizing stool samples was critical for metabolomic analysis, but not necessary for microbiome analysis. Short-term room temperature storage had a minimal effect on the microbiome and metabolome profiles and is recommended for short-term fecal sample storage. In addition, our study indicates that the use of RNALater as a storage medium of stool samples for microbial and metabolomic analysis is not recommended.