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 |
|---|---|
| Pages (from-to) | 464-467 |
| Number of pages | 4 |
| Journal | Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society |
| Volume | 14 |
| Issue number | 4 |
| Early online date | 24 Oct 2014 |
| DOIs | |
| Publication status | Published - Jul 2015 |
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Keywords
- microbiota
- microbiome
- biobank
- pyrosequencing
- propidium monoazide
- sputum
Cite this
Implications of multiple freeze-thawing on respiratory samples for culture-independent analyses. / Cuthbertson, Leah; Rogers, Geraint B; Walker, Alan W; Oliver, Anna; Hoffman, Lucas R; Carroll, Mary P; Parkhill, Julian; Bruce, Kenneth D; van der Gast, Christopher J.
In: Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society, Vol. 14, No. 4, 07.2015, p. 464-467.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Implications of multiple freeze-thawing on respiratory samples for culture-independent analyses
AU - Cuthbertson, Leah
AU - Rogers, Geraint B
AU - Walker, Alan W
AU - Oliver, Anna
AU - Hoffman, Lucas R
AU - Carroll, Mary P
AU - Parkhill, Julian
AU - Bruce, Kenneth D
AU - van der Gast, Christopher J
N1 - Copyright © 2014. Published by Elsevier B.V. Acknowledgements This study was supported by the UK Natural Environment Research Council (NE/H019456/1) to CJvdG, by the Wellcome Trust (WT 098051) to AWW and JP, and by the US National Institute of Health (K02HL105543 and P30 DK089507) to LRH.
PY - 2015/7
Y1 - 2015/7
N2 - 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.
AB - 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.
KW - microbiota
KW - microbiome
KW - biobank
KW - pyrosequencing
KW - propidium monoazide
KW - sputum
U2 - 10.1016/j.jcf.2014.10.004
DO - 10.1016/j.jcf.2014.10.004
M3 - Article
C2 - 25459563
VL - 14
SP - 464
EP - 467
JO - Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society
JF - Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society
SN - 1873-5010
IS - 4
ER -