Comparison of Collection Methods for Fecal Samples in Microbiome Studies

Abstract

Prospective cohort studies are needed to assess the relationship between the fecal microbiome and human health and disease. To evaluate fecal collection methods, we determined technical reproducibility, stability at ambient temperature, and accuracy of 5 fecal collection methods (no additive, 95% ethanol, RNAlater Stabilization Solution, fecal occult blood test cards, and fecal immunochemical test tubes). Fifty-two healthy volunteers provided fecal samples at the Mayo Clinic in Rochester, Minnesota, in 2014. One set from each sample collection method was frozen immediately, and a second set was incubated at room temperature for 96 hours and then frozen. Intraclass correlation coefficients (ICCs) were calculated for the relative abundance of 3 phyla, 2 alpha diversity metrics, and 4 beta diversity metrics. Technical reproducibility was high, with ICCs for duplicate fecal samples between 0.64 and 1.00. Stability for most methods was generally high, although the ICCs were below 0.60 for 95% ethanol in metrics that were more sensitive to relative abundance. When compared with fecal samples that were frozen immediately, the ICCs were below 0.60 for the metrics that were sensitive to relative abundance; however, the remaining 2 alpha diversity and 3 beta diversity metrics were all relatively accurate, with ICCs above 0.60. In conclusion, all fecal sample collection methods appear relatively reproducible, stable, and accurate. Future studies could use these collection methods for microbiome analyses.

Keywords: feces; microbiota; specimen collection.

Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Figures

Figure 1.

Technical reproducibility of replicates frozen immediately (A) and frozen after incubation at room temperature for 4 days (B) for the evaluation of the relative abundance of 3 phyla, 2 alpha diversity metrics, and the first principal coordinate of 4 beta diversity metrics using intraclass correlation coefficients, Mayo Clinic, Rochester, Minnesota, 2014. White columns indicate replicates stored in no solution, striped columns indicate those stored in fecal immunochemical test tubes, gray columns indicate those stored on fecal occult blood test cards, dotted columns indicate those stored in RNAlater Stabilization Solution (Ambion, Austin, Texas), and black columns indicate those stored in 95% ethanol. BC, Bray-Curtis distance; OTU, operational taxonomic unit; PC1, principal coordinate analysis component 1; SDI, Shannon diversity index.

Figure 2.

Stability of fecal samples incubated at room temperature for 4 days and then frozen compared with that of samples frozen immediately for the evaluation of relative abundance of 3 phyla, 2 alpha diversity metrics, and the first principal coordinate of 4 beta diversity metrics using intraclass correlation coefficients, Mayo Clinic, Rochester, Minnesota, 2014. Striped columns indicate those stored in fecal immunochemical test tubes, gray columns indicate those stored on fecal occult blood test cards, dotted columns indicate those stored in RNAlater Stabilization Solution (Ambion, Austin, Texas), and black columns indicate those stored in 95% ethanol. BC, Bray-Curtis distance; OTU, operational taxonomic unit; PC1, principal coordinate analysis component 1; SDI, Shannon diversity index.

Figure 3.

Accuracy of fecal samples frozen immediately compared with the gold standard, which was considered to be the no-additive sample, Mayo Clinic, Rochester, Minnesota, 2014. Intraclass correlation coefficients (A) and Spearman correlation coefficients (B) were calculated for the relative abundance of 3 phyla, 2 alpha diversity metrics, and the first principal coordinate of 4 beta diversity metrics. Striped columns indicate those stored in fecal immunochemical test tubes, gray columns indicate those stored on fecal occult blood test cards, dotted columns indicate those stored in RNAlater Stabilization Solution (Ambion, Austin, Texas), and black columns indicate those stored in 95% ethanol. BC, Bray-Curtis distance; OTU, operational taxonomic unit; PC1, principal coordinate analysis component 1; SDI, Shannon diversity index.