Evaluating stool microbiome integrity after domestic freezer storage using whole-metagenome sequencing, genome assembly, and antimicrobial resistance gene analysis

ABSTRACT The gut microbiome is crucial for host health. Early childhood is a critical period for the development of a healthy gut microbiome, but it is particularly sensitive to external influences. Recent research has focused on using advanced techniques like shotgun metagenome sequencing to identify key microbial signatures and disruptions linked to disease. For accurate microbiome analysis, samples need to be collected and stored under specific conditions to preserve microbial integrity and composition, with −80°C storage considered the gold standard for stabilization. This study investigates the effect of domestic freezer storage on the microbial composition of stool samples obtained from 20 children under 4 years of age with the use of shotgun metagenome sequencing. Fresh stool samples were aliquoted into sterile tubes, with one aliquot stored at 4°C and analyzed within 24 hours, while others were frozen in domestic freezers (below −18°C) and analyzed after 1 week, 2 months, and 6 months. Assessments of contig assembly quality, microbial diversity, and antimicrobial resistance genes revealed no significant degradation or variation in microbial composition.IMPORTANCEMost prior studies on sample storage have relied on amplicon sequencing, which is less applicable to metagenome sequencing—given considerations of contig quality and functional gene detection—and less reliable in representing microbial composition. Moreover, the effects of domestic freezer storage for at-home stool collection on microbiome profiles, contig quality, and antimicrobial resistance gene profiles have not been previously investigated. Our findings suggest that stool samples stored in domestic freezers for up to 6 months maintain the integrity of metagenomic data. These findings indicate that domestic freezer storage does not compromise the integrity or reproducibility of metagenomic data, offering a reliable and accessible alternative for temporary sample storage. This approach enhances the feasibility of large-scale at-home stool collection and citizen science projects, even those focused on the more easily perturbed early life microbiome. This advancement enables more inclusive research into the gut microbiome, enhancing our understanding of its role in human health.