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

Cuthbertson, L, Rogers, GB, Walker, AW, Oliver, A, Hoffman, LR, Carroll, MP, Parkhill, J, Bruce, KD and van der Gast, CJ (2015) Implications of multiple freeze-thawing on respiratory samples for culture-independent analyses. Journal of Cystic Fibrosis, 14 (4). pp. 464-467. ISSN 1569-1993

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Abstract

© 2014 . 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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