Abstract

The development of next-generation sequencing technologies has spurred a surge of research on bacterial microbiome diversity and function. But despite the rapid growth of the field, many uncertainties remain regarding the impact of differing methodologies on downstream results. Sample storage temperature is conventionally thought to be among the most important factors for ensuring reproducibility across marker gene studies, but to date much of the research on this topic has focused on short-term storage in the context of clinical applications. Consequently, it has remained unclear if storage at −80 °C, widely viewed as the gold standard for long-term archival of feces, is truly required for maintaining sample integrity in amplicon-based studies. A better understanding of the impacts of long-term storage conditions is important given the substantial cost and limited availability of ultra-low temperature freezers. To this end, we compared bacterial microbiome profiles inferred from 16S V3–V4 amplicon sequencing for paired fecal samples obtained from a feral horse population from Sable Island, Nova Scotia, Canada, stored at either −80 °C or −20 °C for 4 years. We found that storage temperature did not significantly affect alpha diversity measures, including amplicon sequence variant (ASV) richness and evenness, and abundance of rare sequence variants, nor presence/absence, relative abundances and phylogenetic diversity weighted measures of beta diversity. These results indicate that storage of equine feces at −20 °C for periods ranging from a few months to a few years is equivalent to storage at −80 °C for amplicon-based microbiome studies, adding to accumulating evidence indicating that standard domestic freezers are both economical and effective for microbiome research.

Highlights

  • Targeted marker gene sequencing allows for the characterization of biological communities at high-resolution (Cristescu, 2014)

  • Long-term storage of feces at −80 C versus −20 C is negligible for 16S rRNA amplicon profiling of the equine bacterial microbiome

  • Given the need to validate storage protocols currently in use by our group and others for projects where access to −80 C storage is limited or impossible, we evaluated the effect of long-term freezing storage temperature on fecal samples collected as part of a long-term study of feral horses on Sable Island, Nova Scotia, Canada

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Summary

Introduction

Targeted marker gene sequencing allows for the characterization of biological communities at high-resolution (Cristescu, 2014). The use of different DNA extraction kits and amplification methodologies have been observed to introduce technical variation comparable to that of biological variation in metagenomic and amplicon-based experiments (Costea et al, 2017; Sinha et al, 2017). While these effects are substantial and well-documented, the spread of marker gene surveys has brought with it a renewed interest in optimizing and finding alternative sample storage solutions, which are known as large sources of technical variation (Kim et al, 2017)

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