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

doi:10.1016/j.jcf.2014.10.004

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 journal › Article › peer-review

18 Citations (Scopus)

6 Downloads (Pure)

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 : official journal of the European Cystic Fibrosis Society
Volume	14
Issue number	4
Early online date	24 Oct 2014
DOIs	https://doi.org/10.1016/j.jcf.2014.10.004
Publication status	Published - Jul 2015

Keywords

microbiota
microbiome
biobank
pyrosequencing
propidium monoazide
sputum

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Implications of multiple freeze-thawing on respiratory samples for culture-independent analyses
This is an Open Access article distributed in accordance with the Creative Commons Attribution Non Commercial-No Derivs (CC BY-NC-ND 3.0) license, which permits others to distribute this work non-commercially provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc-nd/3.0/
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Cuthbertson, L., Rogers, G. B., Walker, A. W., Oliver, A., Hoffman, L. R., Carroll, M. P., Parkhill, J., Bruce, K. D., & van der Gast, C. J. (2015). Implications of multiple freeze-thawing on respiratory samples for culture-independent analyses. Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society, 14(4), 464-467. https://doi.org/10.1016/j.jcf.2014.10.004

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 journal › Article › peer-review

Cuthbertson, L, Rogers, GB, Walker, AW, Oliver, A, Hoffman, LR, Carroll, MP, Parkhill, J, Bruce, KD & van der Gast, CJ 2015, 'Implications of multiple freeze-thawing on respiratory samples for culture-independent analyses', Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society, vol. 14, no. 4, pp. 464-467. https://doi.org/10.1016/j.jcf.2014.10.004

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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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.",

keywords = "microbiota, microbiome, biobank, pyrosequencing, propidium monoazide, sputum",

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

note = "Copyright {\textcopyright} 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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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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N2 - 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.

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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