The importance of defining whole stool, sterile sampling methods and homogenization procedures for gut microbiome characterization

Authors

• SE Mudra
• J Barb
• K Schuebel
• S Shim
• S Klagholz
• GR Wallen
• NJ Ames

Abstract

Although gut microbiome characterization has dramatically improved following next generation sequencing, sampling methods are rarely highlighted in manuscripts and consensus does not exist regarding best sampling practices—a significant limitation for downstream analysis and cross-study comparison. Studies indicate taxonomic variance by stool sampling site; consequently, we attempt to diminish bias trough a novel, whole stool homogenization procedure. Methods sought to maximize sterility, feasibility and microbial representation. Entire stool specimens were collected from patients undergoing 28-day inpatient alcohol detoxification at the NIH Clinical Center. Specimens were frozen at -20 °C within 15 minutes of defecation. Within three days, samples were thawed, homogenized, and frozen at -80 °C prior to DNA extraction, library construction and sequencing. DNA extraction used the MagAttract PowerMicrobiome RNA/DNA isolation kit, optimized for KingFisher Duo Prime. 16S rRNA amplification used the Ion 16S Metagenomics Kit through multiplexed primer sets V2, V4, V8 and V3, V67, V9. Amplicons were sequenced with the Ion Torrent semiconductor sequencer and Hi-Q View sequencing reagents with the Ion 318 v2 chip. Data analysis employed Mothur, QIIME and UPARSE with Greengenes as the reference. Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria were highly represented—consistent with the American Gut Project. These preliminary results highlight the utility of whole stool homogenization to capture the gut microbiome and begin to address the challenge of stool subsampling. This project was funded through the joint support of the Clinical Center Nursing Department and the National Institute on Alcohol Abuse and Alcoholism.

Scientific Focus Area: Microbiology and Infectious Diseases

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