Implications of multiple freeze-thawing on respiratory samples for culture-independent analyses

Leah Cuthbertson, Geraint B Rogers, Alan W Walker, Anna Oliver, Lucas R Hoffman, Mary P Carroll, Julian Parkhill, Kenneth D Bruce, Christopher J van der Gast

Research output: Contribution to journalArticle

14 Citations (Scopus)
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Abstract

BACKGROUND: Best practice when performing culture-independent microbiological analysis of sputum samples involves their rapid freezing and storage at -80°C. However, accessing biobanked collections can mean that material has been passed through repeated freeze-thaw cycles. The aim of this study was to determine the impact of these cycles on microbial community profiles.

METHODS: Sputum was collected from eight adults with cystic fibrosis, and each sample was subjected to six freeze-thaw cycles. Following each cycle, an aliquot was removed and treated with propidium monoazide (PMA) prior to DNA extraction and 16S rRNA gene pyrosequencing.

RESULTS: The impact of freeze-thaw cycles was greatest on rare members of the microbiota, with variation beyond that detected with within-sample repeat analysis observed after three cycles.

CONCLUSION: Four or more freeze thaw cycles result in a significant distortion of microbiota profiles from CF sputum.

Original languageEnglish
Pages (from-to)464-467
Number of pages4
JournalJournal of cystic fibrosis : official journal of the European Cystic Fibrosis Society
Volume14
Issue number4
Early online date24 Oct 2014
DOIs
Publication statusPublished - Jul 2015

Fingerprint

Sputum
Microbiota
rRNA Genes
Practice Guidelines
Cystic Fibrosis
Freezing
DNA

Keywords

  • microbiota
  • microbiome
  • biobank
  • pyrosequencing
  • propidium monoazide
  • sputum

Cite this

Implications of multiple freeze-thawing on respiratory samples for culture-independent analyses. / Cuthbertson, Leah; Rogers, Geraint B; Walker, Alan W; Oliver, Anna; Hoffman, Lucas R; Carroll, Mary P; Parkhill, Julian; Bruce, Kenneth D; van der Gast, Christopher J.

In: Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society, Vol. 14, No. 4, 07.2015, p. 464-467.

Research output: Contribution to journalArticle

Cuthbertson, Leah ; Rogers, Geraint B ; Walker, Alan W ; Oliver, Anna ; Hoffman, Lucas R ; Carroll, Mary P ; Parkhill, Julian ; Bruce, Kenneth D ; van der Gast, Christopher J. / Implications of multiple freeze-thawing on respiratory samples for culture-independent analyses. In: Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society. 2015 ; Vol. 14, No. 4. pp. 464-467.
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abstract = "BACKGROUND: Best practice when performing culture-independent microbiological analysis of sputum samples involves their rapid freezing and storage at -80°C. However, accessing biobanked collections can mean that material has been passed through repeated freeze-thaw cycles. The aim of this study was to determine the impact of these cycles on microbial community profiles.METHODS: Sputum was collected from eight adults with cystic fibrosis, and each sample was subjected to six freeze-thaw cycles. Following each cycle, an aliquot was removed and treated with propidium monoazide (PMA) prior to DNA extraction and 16S rRNA gene pyrosequencing.RESULTS: The impact of freeze-thaw cycles was greatest on rare members of the microbiota, with variation beyond that detected with within-sample repeat analysis observed after three cycles.CONCLUSION: Four or more freeze thaw cycles result in a significant distortion of microbiota profiles from CF sputum.",
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AU - Hoffman, Lucas R

AU - Carroll, Mary P

AU - Parkhill, Julian

AU - Bruce, Kenneth D

AU - van der Gast, Christopher J

N1 - Copyright © 2014. Published by Elsevier B.V. Acknowledgements This study was supported by the UK Natural Environment Research Council (NE/H019456/1) to CJvdG, by the Wellcome Trust (WT 098051) to AWW and JP, and by the US National Institute of Health (K02HL105543 and P30 DK089507) to LRH.

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AB - BACKGROUND: Best practice when performing culture-independent microbiological analysis of sputum samples involves their rapid freezing and storage at -80°C. However, accessing biobanked collections can mean that material has been passed through repeated freeze-thaw cycles. The aim of this study was to determine the impact of these cycles on microbial community profiles.METHODS: Sputum was collected from eight adults with cystic fibrosis, and each sample was subjected to six freeze-thaw cycles. Following each cycle, an aliquot was removed and treated with propidium monoazide (PMA) prior to DNA extraction and 16S rRNA gene pyrosequencing.RESULTS: The impact of freeze-thaw cycles was greatest on rare members of the microbiota, with variation beyond that detected with within-sample repeat analysis observed after three cycles.CONCLUSION: Four or more freeze thaw cycles result in a significant distortion of microbiota profiles from CF sputum.

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