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Home / Equine Research Bank / PeerJ / Long-term storage of feces at -80 °C versus -20 °C...
PeerJ2021; 9; e10837; doi: 10.7717/peerj.10837

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

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.
© 2021 Gavriliuc et al.
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Publication Date: 2021-03-09 PubMed ID: 33854827PubMed Central: PMC7953882DOI: 10.7717/peerj.10837Google Scholar: Lookup
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  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research article evaluated the effects of the storage temperature of fecal samples on the accuracy of bacterial microbiome profiling. The authors found no significant difference between storing samples at -80°C and -20°C for four years from horse population, demonstrating that domestic freezers can effectively perform this function.

Study Objective and Importance

  • The study aimed to determine the impact of long-term storage temperatures on the reproductive accuracy in amplicon-based microbiome studies. The importance of this research is due to the higher cost and limited availability of ultra-low freezers, often considered the gold standard for long-term storage of fecal samples.

Research Methodology

  • To achieve this, researchers measured bacterial microbiome profiles from paired fecal samples using 16S V3-V4 amplicon sequencing. This sequencing method is widely used to identify bacteria and other organisms present in a biological sample.
  • The fecal samples used in the study came from a feral horse population in Sable Island, Nova Scotia, Canada. The samples were stored for four years under two different temperature conditions: -80°C and -20°C.

Findings

  • According to the research findings, storage temperature does not have a significant impact on alpha diversity measures. This includes amplicon sequence variant (ASV) richness and evenness, and the abundance of rare sequence variants.
  • Similarly, the storage temperatures don’t significantly influence presence/absence, relative abundances, and phylogenetic diversity weighted measures of beta diversity.

Conclusion and Implications

  • The study concludes that storage of equine feces at -20°C for periods ranging from few months to a few years yields comparable results to storage at -80°C, for amplicon-based microbiome studies.
  • Hence, it suggests that standard domestic freezers could be economical and effective alternatives for microbiome research, a finding that holds potential for large-scale, resource-limited applications.

Cite This Article

APA
Gavriliuc S, Stothart MR, Henry A, Poissant J. (2021). Long-term storage of feces at -80 °C versus -20 °C is negligible for 16S rRNA amplicon profiling of the equine bacterial microbiome. PeerJ, 9, e10837. https://doi.org/10.7717/peerj.10837

Publication

PeerJ
ISSN: 2167-8359
NlmUniqueID: 101603425
Country: United States
Language: English
Volume: 9
Pages: e10837
PII: e10837

Researcher Affiliations

Gavriliuc, Stefan
  • Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada.
Stothart, Mason R
  • Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada.
Henry, Astrid
  • Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada.
Poissant, Jocelyn
  • Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada.

Conflict of Interest Statement

The authors declare that they have no competing interests.

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Citations

This article has been cited 3 times.
  1. Kiet BT, Nhung NT, Yen NTP, Phu DH, Dung NTT, Yen LK, Thu HTV, Carrique-Mas JJ. Impact of Freeze Storage on the Estimation of Phenotypic Antimicrobial Resistance Prevalence in Escherichia coli Collected from Faecal Samples from Healthy Humans and Chickens.. Antibiotics (Basel) 2022 Nov 17;11(11).
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  2. Bensch HM, Tolf C, Waldenström J, Lundin D, Zöttl M. Freeze-drying can replace cold-chains for transport and storage of fecal microbiome samples.. PeerJ 2022;10:e13095.
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  3. Shi J, Yang Y, Xu W, Cai H, Wu J, Long J, Cai Q, Zheng W, Flynn CR, Shu XO, Yu D. Sex-Specific Associations between Gut Microbiome and Non-Alcoholic Fatty Liver Disease among Urban Chinese Adults.. Microorganisms 2021 Oct 8;9(10).
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