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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.. 2015 Implications of multiple freeze-thawing on respiratory samples for culture-independent analyses. Journal of Cystic Fibrosis, 14 (4). 464-467. 10.1016/j.jcf.2014.10.004

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Abstract/Summary

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.

Item Type: Publication - Article
Digital Object Identifier (DOI): 10.1016/j.jcf.2014.10.004
CEH Sections: Acreman
ISSN: 1569-1993
Additional Information. Not used in RCUK Gateway to Research.: Open Access paper - Official URL link provides full text
Additional Keywords: microbiota, microbiome, biobank, pyrosequencing, propidium monoazide, sputum
NORA Subject Terms: Health
Medicine
Biology and Microbiology
Date made live: 19 Jan 2015 13:47 +0 (UTC)
URI: http://nora.nerc.ac.uk/id/eprint/509282

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