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.
|Number of pages||4|
|Journal||Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society|
|Early online date||24 Oct 2014|
|Publication status||Published - Jul 2015|
- propidium monoazide