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 language | English |
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Pages (from-to) | 464-467 |
Number of pages | 4 |
Journal | Journal of Cystic Fibrosis |
Volume | 14 |
Issue number | 4 |
DOIs | |
Publication status | Published - 1 Jul 2015 |
Keywords
- Biobank
- Microbiome
- Microbiota
- Propidium monoazide
- Pyrosequencing
- Sputum