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

TitleComparison of Methods To Collect Fecal Samples for Microbiome Studies Using Whole-Genome Shotgun Metagenomic Sequencing.
Publication TypeJournal Article
Year of Publication2020
AuthorsByrd, DA, Sinha, R, Hoffman, KL, Chen, J, Hua, X, Shi, J, Chia, N, Petrosino, J, Vogtmann, E
Date Published2020 Feb 26
KeywordsAdult, DNA, Bacterial, Ethanol, Feces, Female, Freezing, Gastrointestinal Microbiome, Healthy Volunteers, Humans, Male, Metagenome, Metagenomics, Middle Aged, Preservation, Biological, RNA, Ribosomal, 16S, Specimen Handling, Temperature, Whole Genome Sequencing

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. 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.

Alternate JournalmSphere
PubMed ID32250964
PubMed Central IDPMC7045388
Grant ListR01 CA179243 / CA / NCI NIH HHS / United States

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