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    The Effects of Freezing on Faecal Microbiota as Determined Using MiSeq Sequencing and Culture-Based Investigations

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    Author
    Fouhy, Fiona cc
    Deane, Jennifer
    Rea, Mary cc
    O'Sullivan, Orla cc
    Ross, R Paul
    O'Callaghan, Grace
    Plant, Barry J.
    STANTON, CATHERINE cc
    Keyword
    Gut bacteria
    Specimen storage
    DNA extraction
    Date
    06/03/2015
    
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    URI
    http://hdl.handle.net/11019/821
    Citation
    Fouhy F, Deane J, Rea MC, O’Sullivan Ó, Ross RP, O’Callaghan G, et al. (2015) The Effects of Freezing on Faecal Microbiota as Determined Using MiSeq Sequencing and Culture-Based Investigations. PLoS ONE 10(3): e0119355. doi:10.1371/journal.pone.0119355
    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.
    Funder
    Science Foundation Ireland; European Union
    Grant Number
    02/CE/B124; 603038; SFI/12/RC/2273
    ae974a485f413a2113503eed53cd6c53
    http://dx.doi.org/10.1371/journal.pone.0119355
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    Food Biosciences
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