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 journalArticlepeer-review

20 Citations (Scopus)
7 Downloads (Pure)


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 : official journal of the European Cystic Fibrosis Society
Issue number4
Early online date24 Oct 2014
Publication statusPublished - Jul 2015


  • microbiota
  • microbiome
  • biobank
  • pyrosequencing
  • propidium monoazide
  • sputum


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