Reproducibility, stability, and accuracy of microbial profiles by fecal sample collection method in three distinct populations

• Published in: PloS one Vol. 14; no. 11; p. e0224757
• Main Authors: Byrd, Doratha A., Chen, Jun, Vogtmann, Emily, Hullings, Autumn, Song, Se Jin, Amir, Amnon, Kibriya, Muhammad G., Ahsan, Habibul, Chen, Yu, Nelson, Heidi, Knight, Rob, Shi, Jianxin, Chia, Nicholas, Sinha, Rashmi
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 18.11.2019; Public Library of Science (PLoS)
• Subjects: Ambient temperature; Analysis; Biodiversity; Biology and Life Sciences; Biomarkers; Blood tests; Collection; Colorectal cancer; Correlation; Correlation coefficient; Correlation coefficients; Deoxyribonucleic acid; DNA; DNA, Bacterial - genetics; DNA, Bacterial - isolation & purification; Ecology and Environmental Sciences; Epidemiology; Ethanol; Etiology; Etiology (Medicine); Feasibility Studies; Fecal microflora; Fecal occult blood test; Feces; Feces - microbiology; Gastrointestinal Microbiome - genetics; Gastrointestinal system; Gastrointestinal tract; Gene sequencing; Genes; Gorillas; Health; Health sciences; Healthy Volunteers; Humans; Immunochemistry - instrumentation; Immunochemistry - methods; Intestinal microflora; Laboratories; Medical tests; Medicine and Health Sciences; Metabolism; Metabolites; Methods; Microbiomes; Microbiota; Microorganisms; Pediatrics; People and Places; Physical Sciences; Populations; Precision medicine; Public health; Relative abundance; Reproducibility; Reproducibility of Results; Research and Analysis Methods; RNA; RNA, Ribosomal, 16S - genetics; rRNA 16S; Sampling methods; Specimen Handling - instrumentation; Specimen Handling - methods; Specimen Handling - standards; Squamous cell carcinoma; Stability; Studies; Technology; Tubes; Bangladesh; Colorado; La Jolla California; New York; United States > US; Illinois; Minnesota; California; Chicago Illinois; Maryland
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0224757

The gut microbiome likely plays a role in the etiology of multiple health conditions, especially those affecting the gastrointestinal tract. Little consensus exists as to the best, standard methods to collect fecal samples for future microbiome analysis. We evaluated three distinct populations (N = 132 participants) using 16S rRNA gene amplicon sequencing data to investigate the reproducibility, stability, and accuracy of microbial profiles in fecal samples collected and stored via fecal occult blood test (FOBT) or Flinders Technology Associates (FTA) cards, fecal immunochemical tests (FIT) tubes, 70% and 95% ethanol, RNAlater, or with no solution. For each collection method, based on relative abundance of select phyla and genera, two alpha diversity metrics, and four beta diversity metrics, we calculated intraclass correlation coefficients (ICCs) to estimate reproducibility and stability, and Spearman correlation coefficients (SCCs) to estimate accuracy of the fecal microbial profile. Comparing duplicate samples, reproducibility ICCs for all collection methods were excellent (ICCs ≥75%). After 4-7 days at ambient temperature, ICCs for microbial profile stability were excellent (≥75%) for most collection methods, except those collected via no-solution and 70% ethanol. SCCs comparing each collection method to immediately-frozen no-solution samples ranged from fair to excellent for most methods; however, accuracy of genus-level relative abundances differed by collection method. Our findings, taken together with previous studies and feasibility considerations, indicated that FOBT/FTA cards, FIT tubes, 95% ethanol, and RNAlater are excellent choices for fecal sample collection methods in future microbiome studies. Furthermore, establishing standard collection methods across studies is highly desirable.