Participants with BMI < 95th percentile

Participants with BMI > or = 95th percentile

High-throughput sequencing targeting the 16S rRNA gene will be used to detect and identify bacteria in fecal samples. DNA extraction and library preparation will be performed at the University of Illinois College of Medicine at Peoria, and libraries will be subjected to 16S rDNA sequencing (approximately 10k reads will be obtained per sample) on a MiSeq instrument. Using 10k reads per sample provides stable patterns for human stool microbiota. The 16S data will provide information about the presence and abundance of bacteria, and will be analyzed with Qiime, an open source bioinformatics package.

Gas Chromatography will be used to measure SCFA amounts, specifically acetate, butyrate, proprionate. Preserved aliquot samples will be analyzed on Agilent GC/MS system (6890GC/5973 MS with 7683B autosampler) with gas chromatography performed on a ZB-WAX (30 m×0.32 mm I.D., 0.25um film thickness) capillary column (Phenomenex, Torrance, CA). The inlet and MSD interface temperatures will both be 250°C, with the ion source temperature adjusted to 230°C. The helium carrier gas will be kept at a constant flow rate of 3.2 ml/min. The temperature program is: initial 2-min isothermal heating at 80°C, followed by an oven temperature increase of 20°C/min to 110°C, then 10°C/min to 130°C, and then 35°C/min to 255°C. Mass spectra will again be recorded in the m/z 50-300 scanning range.

Technical factors, which may affect microbial compositions (such as times of specimen collection, home freezer storage removal, shipping, and arrival) will be recorded as independent variables. To estimate and minimize the effect of technical variables, principal component analysis will be used and the study will follow the International Human Microbiome Standards