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. feces; microbiota; specimen collection Abbreviations: FIT, fecal immunochemical test, FOBT, fecal occult blood test, ICC, intraclass correlation coefficient, OTU, operational taxonomic unit.

Comparison of Fecal Collection Methods for Microbiota Studies in Bangladesh

“…In general, our findings agree with those from many of the previous studies considering the impact of the collection method and short-term storage at room temperature in predominantly Western populations (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). In general, FOBT cards or Whatman FTA cards, which have feces smeared on the card itself that then dries, have been found to be relatively stable at room temperature and similar to immediately frozen samples without preservatives (6,10,15,21,22).…”

“…Fecal samples stored in 95% ethanol (12,21) and RNAlater (6,11,12,15,19,22) appear to be adequately preserved in most studies, although some studies found some decreased diversity, DNA quality, or stability in samples stored in RNAlater (9,10,13,19,21). We and another group previously observed that FIT tubes, which store a small amount of feces from a probe in a buffer solution, stabilize fecal samples (22,23), but we were unable to replicate this finding in this study in Bangladesh.…”

“…In general, FOBT cards or Whatman FTA cards, which have feces smeared on the card itself that then dries, have been found to be relatively stable at room temperature and similar to immediately frozen samples without preservatives (6,10,15,21,22). Fecal samples stored in 95% ethanol (12,21) and RNAlater (6,11,12,15,19,22) appear to be adequately preserved in most studies, although some studies found some decreased diversity, DNA quality, or stability in samples stored in RNAlater (9,10,13,19,21). We and another group previously observed that FIT tubes, which store a small amount of feces from a probe in a buffer solution, stabilize fecal samples (22,23), but we were unable to replicate this finding in this study in Bangladesh.…”

“…It is unknown whether cohort participants who refused to provide a fecal specimen had different microbial compositions that might have affected our measurements, although this is unlikely. In addition, the fecal samples were shipped from Bangladesh to the National Cancer Institute and to the University of California, San Diego, and may have experienced some thawing in transit, which might have been related to some of our observed decreases in technical reproducibility, stability, and accuracy compared with those from our previous studies (6,22). We also only investigated 16S rRNA gene sequencing, and the stability and accuracy may differ for other technologies, such as whole-genome shotgun metagenomics and metabolomics.…”

“…The impact of the type of fecal sample collection method on microbial data has been considered in a number of previous studies (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). However, the majority of these studies were conducted in North American or European populations near academic or clinical laboratories and typically included only a small number of methods.…”