Format

Send to

Choose Destination
PLoS One. 2015 Mar 6;10(3):e0119355. doi: 10.1371/journal.pone.0119355. eCollection 2015.

The effects of freezing on faecal microbiota as determined using MiSeq sequencing and culture-based investigations.

Author information

1
Teagasc Food Research Centre, Moorepark, Fermoy, County Cork, Ireland.
2
Teagasc Food Research Centre, Moorepark, Fermoy, County Cork, Ireland; HRB Clinical Research Facility, University College Cork, Cork, Ireland; School of Microbiology, University College Cork, Cork, Ireland.
3
Teagasc Food Research Centre, Moorepark, Fermoy, County Cork, Ireland; Alimentary Pharmabiotic Centre, Cork, Ireland.
4
College of Science, Engineering and Food Science (SEFS), University College Cork, Cork, Ireland.
5
HRB Clinical Research Facility, University College Cork, Cork, Ireland; Alimentary Pharmabiotic Centre, Cork, Ireland; Cork Adult Cystic Fibrosis Centre, University College Cork, Cork University Hospital, Wilton, Cork, Ireland.

Abstract

BACKGROUND:

High-throughput sequencing has enabled detailed insights into complex microbial environments, including the human gut microbiota. The accuracy of the sequencing data however, is reliant upon appropriate storage of the samples prior to DNA extraction. The aim of this study was to conduct the first MiSeq sequencing investigation into the effects of faecal storage on the microbiota, compared to fresh samples. Culture-based analysis was also completed.

METHODS:

Seven faecal samples were collected from healthy adults. Samples were separated into fresh (DNA extracted immediately), snap frozen on dry ice and frozen for 7 days at -80°C prior to DNA extraction or samples frozen at -80°C for 7 days before DNA extraction. Sequencing was completed on the Illumina MiSeq platform. Culturing of total aerobes, anaerobes and bifidobacteria was also completed.

RESULTS:

No significant differences at phylum or family levels between the treatment groups occurred. At genus level only Faecalibacterium and Leuconostoc were significantly different in the fresh samples compared to the snap frozen group (p = 0.0298; p = 0.0330 respectively). Diversity analysis indicated that samples clustered based on the individual donor, rather than by storage group. No significant differences occurred in the culture-based analysis between the fresh, snap or -80°C frozen samples.

CONCLUSIONS:

Using the MiSeq platform coupled with culture-based analysis, this study highlighted that limited significant changes in microbiota occur following rapid freezing of faecal samples prior to DNA extraction. Thus, rapid freezing of samples prior to DNA extraction and culturing, preserves the integrity of the microbiota.

PMID:
25748176
PMCID:
PMC4352061
DOI:
10.1371/journal.pone.0119355
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Public Library of Science Icon for PubMed Central
Loading ...
Support Center