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Research Article | Applied and Environmental Science

Preservation Methods Differ in Fecal Microbiome Stability, Affecting Suitability for Field Studies

Se Jin Song, Amnon Amir, Jessica L. Metcalf, Katherine R. Amato, Zhenjiang Zech Xu, Greg Humphrey, Rob Knight
M. Denise Dearing, Editor
Se Jin Song
aDepartment of Pediatrics, University of California—San Diego, La Jolla, California, USA
bDepartment of Ecology and Evolutionary Biology, University of Colorado—Boulder, Boulder, Colorado, USA
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Amnon Amir
aDepartment of Pediatrics, University of California—San Diego, La Jolla, California, USA
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Jessica L. Metcalf
aDepartment of Pediatrics, University of California—San Diego, La Jolla, California, USA
bDepartment of Ecology and Evolutionary Biology, University of Colorado—Boulder, Boulder, Colorado, USA
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Katherine R. Amato
cDepartment of Anthropology, Northwestern University, Evanston, Illinois, USA
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Zhenjiang Zech Xu
aDepartment of Pediatrics, University of California—San Diego, La Jolla, California, USA
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Greg Humphrey
aDepartment of Pediatrics, University of California—San Diego, La Jolla, California, USA
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Rob Knight
aDepartment of Pediatrics, University of California—San Diego, La Jolla, California, USA
dDepartment of Computer Science and Engineering, University of California—San Diego, La Jolla, California, USA
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M. Denise Dearing
University of Utah
Roles: Editor
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DOI: 10.1128/mSystems.00021-16
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ABSTRACT

Immediate freezing at −20°C or below has been considered the gold standard for microbiome preservation, yet this approach is not feasible for many field studies, ranging from anthropology to wildlife conservation. Here we tested five methods for preserving human and dog fecal specimens for periods of up to 8 weeks, including such types of variation as freeze-thaw cycles and the high temperature fluctuations often encountered under field conditions. We found that three of the methods—95% ethanol, FTA cards, and the OMNIgene Gut kit—can preserve samples sufficiently well at ambient temperatures such that differences at 8 weeks are comparable to differences among technical replicates. However, even the worst methods, including those with no fixative, were able to reveal microbiome differences between species at 8 weeks and between individuals after a week, allowing meta-analyses of samples collected using various methods when the effect of interest is expected to be larger than interindividual variation (although use of a single method within a study is strongly recommended to reduce batch effects). Encouragingly for FTA cards, the differences caused by this method are systematic and can be detrended. As in other studies, we strongly caution against the use of 70% ethanol. The results, spanning 15 individuals and over 1,200 samples, provide our most comprehensive view to date of storage effects on stool and provide a paradigm for the future studies of other sample types that will be required to provide a global view of microbial diversity and its interaction among humans, animals, and the environment.

IMPORTANCE Our study, spanning 15 individuals and over 1,200 samples, provides our most comprehensive view to date of storage and stabilization effects on stool. We tested five methods for preserving human and dog fecal specimens for periods of up to 8 weeks, including the types of variation often encountered under field conditions, such as freeze-thaw cycles and high temperature fluctuations. We show that several cost-effective methods provide excellent microbiome stability out to 8 weeks, opening up a range of field studies with humans and wildlife that would otherwise be cost-prohibitive.

  • Copyright © 2016 Song et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license .

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Preservation Methods Differ in Fecal Microbiome Stability, Affecting Suitability for Field Studies
Se Jin Song, Amnon Amir, Jessica L. Metcalf, Katherine R. Amato, Zhenjiang Zech Xu, Greg Humphrey, Rob Knight
mSystems May 2016, 1 (3) e00021-16; DOI: 10.1128/mSystems.00021-16

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Preservation Methods Differ in Fecal Microbiome Stability, Affecting Suitability for Field Studies
Se Jin Song, Amnon Amir, Jessica L. Metcalf, Katherine R. Amato, Zhenjiang Zech Xu, Greg Humphrey, Rob Knight
mSystems May 2016, 1 (3) e00021-16; DOI: 10.1128/mSystems.00021-16
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KEYWORDS

DNA stability
fecal microbiome
sample storage

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