USDA-ARS Cross-Sectional Nutritional Phenotyping Study Gut Microbiota

Researchers from the United States Department of Agriculture (USDA) Western Human Nutrition Research Center conducted a cross-sectional "metabolic phenotyping" study of healthy people in a multi-ethnic cohort balanced for age (18–65 years), sex, and BMI (18.5–45 kg/m2). Details on study endpoints are available at ClinicalTrials.gov NCT02367287. A single stool sample was collected from 365 participants, transported to the Center on ice, homogenized and frozen at -80 C until DNA extraction. Bacterial DNA was extracted from 100mg of stool using the ZymoBIOMICS DNA Miniprep Kit. Samples with low DNA yield or quality were re-extracted using a lower sample volume (50 – 20 mg) to reduce inhibitor content. Extracted DNA was submitted to Dalhousie University Integrated Microbiome Resource for amplification of the 16S rRNA V4 – V5 region (using primers 515F, GTGYCAGCMGCCGCGGTAA, and 926R, CCGYCAATTYMTTTRAGTTT) and sequencing by Illumina MiSeq with 300 bp paired-end V3 chemistry. Sequencing was performed in three batches. Samples that returned a low number of quality reads were resubmitted in subsequent batches.