Not All Is Lost: Resilience of Microbiome Samples to Freezer Failures and Long-term Storage

Advances in technology have facilitated extensive sample collection to explore microbiomes across diverse systems, leading to a growing reliance on ultracold freezers for storing both samples and extracted DNA. However, freezer malfunctions can jeopardize data integrity. In this study, we investigated the effects of an unforeseen short-term thaw event ([~]1 week) resulting from a freezer malfunction on soil samples stored at -80{degrees}C and extracted uncompromised DNA stored long-term at -20{degrees}C. We compared these samples against previously sequenced Illumina MiSeq data to assess whether the process of thawing soil or multi-year extracted DNA storage affected the resilience of bacterial or fungal richness or community composition, thereby impacting our ability to accurately determine treatment effects. Our results reveal substantial resilience in fungal richness and both bacterial and fungal beta-diversity to soil sample thawing and extended frozen DNA storage. This resilience facilitated biological inferences that closely mirrored those observed in the original study. Notably, fungi exhibited greater resilience to short-term thawing compared to bacteria, which showed sensitivity to both thawing and long-term freezing. Moreover, taxonomic composition analysis revealed the persistence of dominant microbial taxa under thawing and prolonged freezing, suggesting that dominant microbes remain viable for tracking across temporal studies. In conclusion, our study highlights that beta-diversity is more robust than alpha-diversity and fungi are more resilient than bacteria. Furthermore, our findings underscore the effectiveness of soil storage at -80{degrees}C compared to storage of extracted DNA at -20{degrees}C, despite potential freezer failures, as the most robust method for long-term storage in microbiome studies.