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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Official URL: https://www.cysticfibrosisjournal.com/article/S156...

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.

Item Type:	Article
Peer-reviewed:	Yes
Date Deposited:	04 Jul 2018 15:06
Publisher:	Elsevier
Additional Information:	This is an Open Access article published in Journal of Cystic Fibrosis, published by Elsevier B.V. on behalf of European Cystic Fibrosis Society, copyright The Author(s).
Divisions:	Faculties > Science and Engineering
Subject terms:	Science & Technology, Life Sciences & Biomedicine, Respiratory System, Microbiota, Microbiome, Biobank, Pyrosequencing, Propidium monoazide, Sputum, CYSTIC-FIBROSIS, INFECTIONS, SPUTUM, Sputum, Humans, Cystic Fibrosis, Specimen Handling, Cryopreservation, Adult, Biological Specimen Banks, Microbiota, Biobank, Microbiome, Microbiota, Propidium monoazide, Pyrosequencing, Sputum, Adult, Biological Specimen Banks, Cryopreservation, Cystic Fibrosis, Humans, Microbiota, Specimen Handling, Sputum, 1103 Clinical Sciences, Respiratory System
URI:	https://e-space.mmu.ac.uk/id/eprint/621048
DOI:	https://doi.org/10.1016/j.jcf.2014.10.004
ISSN	1569-1993

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