Comparing the Metagenomic Performance of Stools Collected from Custom Cards and 95% Ethanol in Epidemiologic Studies Available to Purchase

Stool cards have been used for microbiome assessment in epidemiologic studies.

We compared shotgun metagenomic sequencing from 32 participants who self-collected stool samples from the same bowel movement using a custom stool card versus a collection tube with 95% ethanol fixative in the Nurses’ Health Study II. We evaluated the agreement between methods at both the whole-community and individual species levels. To contextualize the comparison for disease association studies, we assessed the performance of the two collection methods for differentiating colorectal cancer–associated taxa.

Overall, metagenomes from cards and 95% ethanol were highly correlated within individuals. No difference was found in α diversity and only ∼1% of variation in β diversity was explained by the collection method. At the species level, although the relative abundances were highly correlated between card and ethanol sample pairs (Spearman rho = 0.96), 10 (of 239) species showed a differential abundance in paired samples, including overrepresentation of Escherichia coli and underrepresentation of three Streptococcus species in cards compared with ethanol. Among a set of 99 colorectal cancer–associated species, four showed differential abundances between collection methods; however, this number was consistent with that expected by chance.

Metagenomic sequencing using stool samples self-collected using stool cards or 95% ethanol yielded largely consistent results although differential abundances were observed for a small number of individual species.

Stool cards can be a cost-effective alternative to collect stool samples for metagenomic sequencing in epidemiologic studies but warrant additional considerations for data analysis.