Characterization of the fecal microbiota using high-throughput sequencing reveals a stable microbial community during storage

Abstract

The handling and treatment of biological samples is critical when characterizing the composition of the intestinal microbiota between different ecological niches or diseases. Specifically, exposure of fecal samples to room temperature or long term storage in deep freezing conditions may alter the composition of the microbiota. Thus, we stored fecal samples at room temperature and monitored the stability of the microbiota over twenty four hours. We also investigated the stability of the microbiota in fecal samples during a six month storage period at -80°C. As the stability of the fecal microbiota may be affected by intestinal disease, we analyzed two healthy controls and two patients with irritable bowel syndrome (IBS). We used high-throughput pyrosequencing of the 16S rRNA gene to characterize the microbiota in fecal samples stored at room temperature or -80°C at six and seven time points, respectively. The composition of microbial communities in IBS patients and healthy controls were determined and compared using the Quantitative Insights Into Microbial Ecology (QIIME) pipeline. The composition of the microbiota in fecal samples stored for different lengths of time at room temperature or -80°C clustered strongly based on the host each sample originated from. Our data demonstrates that fecal samples exposed to room or deep freezing temperatures for up to twenty four hours and six months, respectively, exhibit a microbial composition and diversity that shares more identity with its host of origin than any other sample.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Schematic of experimental design.

Figure 2. β-diversity analysis of samples.

Principal coordinates analysis (PCoA) of weighted and unweighted UniFrac distances of IBS patient (green and orange triangles) and healthy control (blue squares and red circles) fecal sample aliquots exposed to room temperature and −80°C for different lengths of time (room temperature - 1, 4, 6, 8 and 24 hours; −80°C −1 week and 1, 2, 3, 4, 5 and 6 months). PCoA plots illustrate the subject each sample aliquot originated from (A&E) and the temperature they were stored at (B&F). Average weighted UniFrac distances for all sample aliquots based on storage at room temperature (C&G) or −80°C (D&H) indicate that sample aliquot microbiotas show significantly similarity (*p<0.05).

Figure 3. Abundances of dominant phyla in samples.

Abundances (% of total 16S rRNA sequences) of the predominant bacterial phyla in healthy control and IBS patient fecal sample aliquots exposed to room temperature and −80°C for different lengths of time.