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 journalArticlepeer-review

    19 Citations (Scopus)


    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 languageEnglish
    Pages (from-to)464-467
    Number of pages4
    JournalJournal of Cystic Fibrosis
    Issue number4
    Early online date2015
    Publication statusPublished - 1 Jul 2015


    • Biobank
    • Microbiome
    • Microbiota
    • Propidium monoazide
    • Pyrosequencing
    • Sputum


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