Assessment of the impact of different fecal storage protocols on the microbiota diversity and composition: a pilot study

Abstract

Background: Fecal samples are currently the most commonly studied proxy for gut microbiota. The gold standard of sample handling and storage for microbiota analysis is maintaining the cold chain during sample transfer and immediate storage at - 80 °C. Gut microbiota studies in large-scale, population-based cohorts require a feasible sample collection protocol. We compared the effect of three different storage methods and mock shipment: immediate freezing at - 80 °C, in 95% ethanol stored at room temperature (RT) for 48 h, and on blood collection card stored at RT for 48 h, on the measured composition of fecal microbiota of eight healthy, female volunteers by sequencing the V4 region of the 16S rRNA gene on an Illumina MiSeq.

Results: Shared operational taxonomic units (OTUs) between different methods were 68 and 3% for OTUs > 0.01 and < 0.01% mean relative abundance within each group, respectively. α and β-diversity measures were not significantly impacted by different storage methods. With the exception of Actinobacteria, fecal microbiota profiles at the phylum level were not significantly affected by the storage method. Actinobacteria was significantly higher in samples collected on card compared to immediate freezing (1.6 ± 1.1% vs. 0.4 ± 0.2%, p = 0.005) mainly driven by expansion of Actinobacteria relative abundance in fecal samples stored on card in two individuals. There was no statistically significant difference at lower taxonomic levels tested.

Conclusion: Consistent results of the microbiota composition and structure for different storage methods were observed. Fecal collection on card could be a suitable alternative to immediate freezing for fecal microbiota analysis using 16S rRNA gene amplicon sequencing.

Keywords: Card; Cohort study; Fecal storage; Gut microbiota; Mock shipment.

Fig. 1

Effect of storage condition on the fecal microbiota profile. a Venn diagram of abundant and rare operational taxonomic units (OTUs) defined as having mean relative abundance of > 0.01% and < 0.01% within each method, respectively. b α diversity across methods, and c across individuals. d β diversity based on Bray-Curtis dissimilarity for all and rare OTUs (mean relative abundance < 0.01%)

Fig. 2

Comparison of relative abundances of gut microbiota profile at phylum level a across individuals, and b across methods

Fig. 3

Comparison of relative abundances of abundant genera (> 0.01% mean relative abundance) across individuals and fecal storage methods