Optimized sampling method for fecal microbiome and metabolome preservation under room temperature

Background: The relationship among the human gut microbiome, microbially produced metabolites, and health outcomes remains of great interest. To decrease participant burden, room-temperature storage methods for fecal samples have become increasingly important. However, kits for storing the fecal microbiome and metabolome have not been well explored. We hypothesized that storing fecal samples by drying them with silica gel may be suitable. Objectives: The objective was to evaluate the performance of storage at room temperature by drying feces for subsequent examination of the microbiome, microbial pathways, and the metabolome. Methods: Feces from ten healthy adults (6 male and 4 female) were sampled and immediately processed, as controls, and stored at room temperature in an incubator, on an FTA card, in RNAlater, or dried by silica gel. Storage at room temperature continued for 7 days. Drying by the silica gel method was assessed for 14 days. The fecal microbiome was assessed by sequencing the bacterial 16S ribosomal RNA-encoding gene (V1-V2 region), fecal microbial pathway profiles were analyzed by whole-genome shotgun metagenomics, and fecal metabolome profiles were analyzed using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). Results: Qualitative and β-diversity analyses of the microbiome, microbial pathways, and the metabolome showed that drying by silica gel were closest to those immediately after processing. When focusing on the abundances of individual microbes, microbial pathways, and metabolites, some were found to be significantly different. However, the intra-method ranking of individual items showed that 100%, 87-97%, and 63-76% of microbes, microbial pathways, and metabolites, respectively, were significantly correlated between silica gel preserving and immediately processing method. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study did not receive any funding ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: IRB of Chiyoda Paramedical Care Clinic gave ethical approval for this work I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The obtained sequence data are available in the DNA Data Bank of Japan (DDBJ) Sequence Read Archive (DRA) (DRA accession number: DRA016258).