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Effects of preservation method on canine (Canis lupus familiaris) fecal microbiota

Studies involving gut microbiome analysis play an increasing role in the evaluation of health and disease in humans and animals alike. Fecal sampling methods for DNA preservation in laboratory, clinical, and field settings can greatly influence inferences of microbial composition and diversity, but...

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Autores principales: Horng, Katti R., Ganz, Holly H., Eisen, Jonathan A., Marks, Stanley L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: PeerJ Inc. 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5970549/
https://www.ncbi.nlm.nih.gov/pubmed/29844978
http://dx.doi.org/10.7717/peerj.4827
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author Horng, Katti R.
Ganz, Holly H.
Eisen, Jonathan A.
Marks, Stanley L.
author_facet Horng, Katti R.
Ganz, Holly H.
Eisen, Jonathan A.
Marks, Stanley L.
author_sort Horng, Katti R.
collection PubMed
description Studies involving gut microbiome analysis play an increasing role in the evaluation of health and disease in humans and animals alike. Fecal sampling methods for DNA preservation in laboratory, clinical, and field settings can greatly influence inferences of microbial composition and diversity, but are often inconsistent and under-investigated between studies. Many laboratories have utilized either temperature control or preservation buffers for optimization of DNA preservation, but few studies have evaluated the effects of combining both methods to preserve fecal microbiota. To determine the optimal method for fecal DNA preservation, we collected fecal samples from one canine donor and stored aliquots in RNAlater, 70% ethanol, 50:50 glycerol:PBS, or without buffer at 25 °C, 4 °C, and −80 °C. Fecal DNA was extracted, quantified, and 16S rRNA gene analysis performed on Days 0, 7, 14, and 56 to evaluate changes in DNA concentration, purity, and bacterial diversity and composition over time. We detected overall effects on bacterial community of storage buffer (F-value = 6.87, DF = 3, P < 0.001), storage temperature (F-value=1.77, DF = 3, P = 0.037), and duration of sample storage (F-value = 3.68, DF = 3, P < 0.001). Changes in bacterial composition were observed in samples stored in −80 °C without buffer, a commonly used method for fecal DNA storage, suggesting that simply freezing samples may be suboptimal for bacterial analysis. Fecal preservation with 70% ethanol and RNAlater closely resembled that of fresh samples, though RNAlater yielded significantly lower DNA concentrations (DF = 8.57, P < 0.001). Although bacterial composition varied with temperature and buffer storage, 70% ethanol was the best method for preserving bacterial DNA in canine feces, yielding the highest DNA concentration and minimal changes in bacterial diversity and composition. The differences observed between samples highlight the need to consider optimized post-collection methods in microbiome research.
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spelling pubmed-59705492018-05-29 Effects of preservation method on canine (Canis lupus familiaris) fecal microbiota Horng, Katti R. Ganz, Holly H. Eisen, Jonathan A. Marks, Stanley L. PeerJ Biodiversity Studies involving gut microbiome analysis play an increasing role in the evaluation of health and disease in humans and animals alike. Fecal sampling methods for DNA preservation in laboratory, clinical, and field settings can greatly influence inferences of microbial composition and diversity, but are often inconsistent and under-investigated between studies. Many laboratories have utilized either temperature control or preservation buffers for optimization of DNA preservation, but few studies have evaluated the effects of combining both methods to preserve fecal microbiota. To determine the optimal method for fecal DNA preservation, we collected fecal samples from one canine donor and stored aliquots in RNAlater, 70% ethanol, 50:50 glycerol:PBS, or without buffer at 25 °C, 4 °C, and −80 °C. Fecal DNA was extracted, quantified, and 16S rRNA gene analysis performed on Days 0, 7, 14, and 56 to evaluate changes in DNA concentration, purity, and bacterial diversity and composition over time. We detected overall effects on bacterial community of storage buffer (F-value = 6.87, DF = 3, P < 0.001), storage temperature (F-value=1.77, DF = 3, P = 0.037), and duration of sample storage (F-value = 3.68, DF = 3, P < 0.001). Changes in bacterial composition were observed in samples stored in −80 °C without buffer, a commonly used method for fecal DNA storage, suggesting that simply freezing samples may be suboptimal for bacterial analysis. Fecal preservation with 70% ethanol and RNAlater closely resembled that of fresh samples, though RNAlater yielded significantly lower DNA concentrations (DF = 8.57, P < 0.001). Although bacterial composition varied with temperature and buffer storage, 70% ethanol was the best method for preserving bacterial DNA in canine feces, yielding the highest DNA concentration and minimal changes in bacterial diversity and composition. The differences observed between samples highlight the need to consider optimized post-collection methods in microbiome research. PeerJ Inc. 2018-05-23 /pmc/articles/PMC5970549/ /pubmed/29844978 http://dx.doi.org/10.7717/peerj.4827 Text en ©2018 Horng et al. http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited.
spellingShingle Biodiversity
Horng, Katti R.
Ganz, Holly H.
Eisen, Jonathan A.
Marks, Stanley L.
Effects of preservation method on canine (Canis lupus familiaris) fecal microbiota
title Effects of preservation method on canine (Canis lupus familiaris) fecal microbiota
title_full Effects of preservation method on canine (Canis lupus familiaris) fecal microbiota
title_fullStr Effects of preservation method on canine (Canis lupus familiaris) fecal microbiota
title_full_unstemmed Effects of preservation method on canine (Canis lupus familiaris) fecal microbiota
title_short Effects of preservation method on canine (Canis lupus familiaris) fecal microbiota
title_sort effects of preservation method on canine (canis lupus familiaris) fecal microbiota
topic Biodiversity
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5970549/
https://www.ncbi.nlm.nih.gov/pubmed/29844978
http://dx.doi.org/10.7717/peerj.4827
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