Domestic cryopreservation of fecal samples: an effective strategy for longitudinal studies using multi-omics approaches?

Longitudinal multi-omics alteration of gut microenvironment provided a unique opportunity for the research in intestinal microbiology. However, several questions still remain to be answered, including the subtle shift of multi-omics with different storage temperatures and time courses. A convenient and reliable strategy for fecal sample collection and storage in longitudinal studies is in urgent need.To find out whether the temporal changes of fecal microenvironment profiles could be well preserved at different tempretures, stool samples collected consecutively within a month from 3 healthy volunteers each, were divided into 2 aliquots and stored at different tempretures respectively. A subsequent 16S, ITS1 targeted sequencing and liquid chromatography were implemented to detect fecal bacteria and fungal composition as well as relative bile acids concentration.Our result showed that the cryopreservation at higher tempreture could retain the dynamic alteration patterns of bacterial microbiota within 2 months, suggesting the potentials of using the domestic storage methodology to study fecal microenvironment as well as other possible applications. Of note, a difference of the fungal mycobiota profiles and several microbe-derived bile acids were found between the two cryopreservation temperatures. This indicates further application of the cryopreservation method on fecal fungal mycobiota and parts of metabolites should be taken with prudence.