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

18 Citations (Scopus)

Abstract

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
Volume14
Issue number4
DOIs
Publication statusPublished or Issued - 1 Jul 2015

Keywords

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

ASJC Scopus subject areas

  • Pediatrics, Perinatology, and Child Health
  • Pulmonary and Respiratory Medicine

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