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

BackgroundHigh-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 thisstudy was to conduct the first MiSeq sequencing investigation into the effects of faecal storageon the microbiota, compared to fresh samples. Culture-based analysis wasalso completed.MethodsSeven faecal samples were collected from healthy adults. Samples were separated intofresh (DNA extracted immediately), snap frozen on dry ice and frozen for 7 days at -80°Cprior to DNA extraction or samples frozen at -80°C for 7 days before DNA extraction. Sequencingwas completed on the Illumina MiSeq platform. Culturing of total aerobes, anaerobesand bifidobacteria was also completed.ResultsNo significant differences at phylum or family levels between the treatment groups occurred.At genus level only Faecalibacterium and Leuconostoc were significantly different in thefresh samples compared to the snap frozen group (p = 0.0298; p = 0.0330 respectively). Diversityanalysis indicated that samples clustered based on the individual donor, rather thanby storage group. No significant differences occurred in the culture-based analysis betweenthe fresh, snap or -80°C frozen samples.ConclusionsUsing the MiSeq platform coupled with culture-based analysis, this study highlighted thatlimited significant changes in microbiota occur following rapid freezing of faecal samplesprior to DNA extraction. Thus, rapid freezing of samples prior to DNA extraction and culturing,preserves the integrity of the microbiota.