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

Leah Cuthbertson; Geraint Rogers; Alan Walker; Anna Oliver; Lucas Hoffman; Mary Carroll; Julian Parkhill; Kenneth Bruce; Christopher van der Gast

doi:10.1016/j.jcf.2014.10.004

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

Leah Cuthbertson, Geraint Rogers, Alan Walker, Anna Oliver, Lucas Hoffman, Mary Carroll, Julian Parkhill, Kenneth Bruce, Christopher van der Gast

Research output: Contribution to journal › Article › peer-review

29 Citations (Scopus)

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 language	English
Pages (from-to)	464-467
Number of pages	4
Journal	Journal of Cystic Fibrosis
Volume	14
Issue number	4
Early online date	2015
DOIs	https://doi.org/10.1016/j.jcf.2014.10.004
Publication status	Published - 1 Jul 2015

Keywords

Biobank
Microbiome
Microbiota
Propidium monoazide
Pyrosequencing
Sputum

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10.1016/j.jcf.2014.10.004

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title = "Implications of multiple freeze-thawing on respiratory samples for culture-independent analyses",

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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author = "Leah Cuthbertson and Geraint Rogers and Alan Walker and Anna Oliver and Lucas Hoffman and Mary Carroll and Julian Parkhill and Kenneth Bruce and {van der Gast}, Christopher",

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AU - Cuthbertson, Leah

AU - Rogers, Geraint

AU - Walker, Alan

AU - Oliver, Anna

AU - Hoffman, Lucas

AU - Carroll, Mary

AU - Parkhill, Julian

AU - Bruce, Kenneth

AU - van der Gast, Christopher

PY - 2015/7/1

Y1 - 2015/7/1

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KW - Microbiome

KW - Microbiota

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

Abstract

Keywords

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