The effects of freezing on faecal microbiota as determined using MiSeq sequencing and culture-based investigations.
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.
Teagasc Food Research Centre, Moorepark, Fermoy, County Cork, Ireland.
Fiona
F
Fouhy
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.
Jennifer
J
Deane
Teagasc Food Research Centre, Moorepark, Fermoy, County Cork, Ireland; Alimentary Pharmabiotic Centre, Cork, Ireland.
Mary C
MC
Rea
Teagasc Food Research Centre, Moorepark, Fermoy, County Cork, Ireland; Alimentary Pharmabiotic Centre, Cork, Ireland.
Órla
Ó
O'Sullivan
College of Science, Engineering and Food Science (SEFS), University College Cork, Cork, Ireland.
R Paul
RP
Ross
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.
Grace
G
O'Callaghan
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.
Barry J
BJ
Plant
Teagasc Food Research Centre, Moorepark, Fermoy, County Cork, Ireland; Alimentary Pharmabiotic Centre, Cork, Ireland.
Catherine
C
Stanton
D000328:Adult; D015169:Colony Count, Microbial; D015925:Cryopreservation; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D005243:Feces / Q000382:microbiology*; D005260:Female; D005615:Freezing; D006801:Humans; D007934:Leuconostoc / Q000235:genetics*; D008297:Male; D064307:Microbiota
D004269:DNA, Bacterial