Comparison of Methods To Collect Fecal Samples for Microbiome Studies Using Whole-Genome Shotgun Metagenomic Sequencing

Abstract

Few previous studies have assessed stability and “gold-standard” concordance of fecal sample collection methods for whole-genome shotgun metagenomic sequencing (WGSS), an increasingly popular method for studying the gut microbiome. We used WGSS data to investigate ambient temperature stability and putative gold-standard concordance of microbial profiles in fecal samples collected and stored using fecal occult blood test (FOBT) cards, fecal immunochemical test (FIT) tubes, 95% ethanol, or RNAlater. Among 15 Mayo Clinic employees, for each collection method, we calculated intraclass correlation coefficients (ICCs) to estimate stability of fecal microbial profiles after storage for 4 days at ambient temperature and concordance with immediately frozen, no-solution samples (i.e., the putative gold standard). ICCs were estimated for multiple metrics, including relative abundances of select phyla, species, KEGG k-genes (representing any coding sequence that had >70% identity and >70% query coverage with respect to a known KEGG ortholog), KEGG modules, and KEGG pathways; species and k-gene alpha diversity; and Bray-Curtis and Jaccard species beta diversity. ICCs for microbial profile stability were excellent (≥90%) for fecal samples collected via most of the collection methods, except those preserved in 95% ethanol. Concordance with the immediately frozen, no-solution samples varied for all collection methods, but the number of observed species and the beta diversity metrics tended to have higher concordance than other metrics. Our findings, taken together with previous studies and feasibility considerations, indicated that FOBT cards, FIT tubes, and RNAlater are acceptable choices for fecal sample collection methods in future WGSS studies. IMPORTANCE A major direction for future microbiome research is implementation of fecal sample collections in large-scale, prospective epidemiologic studies. Studying microbiome-disease associations likely requires microbial data to be pooled from multiple studies. Our findings suggest collection methods that are most optimal to be used standardly across future WGSS microbiome studies.

The effects of dairy and dairy derivatives on the gut microbiota: a systematic literature review

“… 39 , 41 , 44 - 46 Moreover, fecal sample collection methods, sample processing methods, and DNA extraction methods influence the output generated. 53 - 55 RNA later stabilizer solution was used for sample collection in one study, 41 and others employed cold-chain collection methods (i.e. no stabilizer).…”

The Maintenance of Microbial Community in Human Fecal Samples by a Self-made Cost-effective Preservation Buffer

“…Interestingly, RNAlater did not constantly inhibit the shift in microbial community after sampling, unable to sustain its well-known role in RNA protection 17,18 . Two medical tools (FOBT and FIT) were also indicated as acceptable choices for fecal sample collection and storage in future sequencing 22 . Apart from these, ethanol has been mentioned most in previous studies to evaluate its impacts on microbial pro les in fecal samples 21,22,30 .…”

“…Nowadays, two major types of preservation agents are available to store sequencing samples, including commercial items and simply self-prepared buffers. To our knowledge, there are approximately a dozen commercial products with certain reputation such as OMNIgene GUT, Norgen, RNAlater, Shield, MGIEasy, Longsee, fecal occult blood test (FOBT) cards, and fecal immunochemical test (FIT) tubes 17,18,22,30 . Among them, OMNIgene GUT kit displays a better performance to stabilize stool samples as multiple teams have reported 17,20 .…”

“…Two medical tools (FOBT and FIT) were also indicated as acceptable choices for fecal sample collection and storage in future sequencing 22 . Apart from these, ethanol has been mentioned most in previous studies to evaluate its impacts on microbial pro les in fecal samples 21,22,30 . More speci cally, 100% ethanol preferably stabilized microbiome structure as compared to 75% ethanol 14,30 .…”

“…Other researchers have acknowledged the bene cial effects of several self-made recipes (e.g. 95% ethanol) on the preservation of fecal samples without altering α-diversity of microbial community 14,21,22 . Nonetheless, the widely-spread application of these agents is still impeded due in part to the inconvenience (e.g.…”