title: 16S rRNA V4 amplicon sequencing of human fecal microbiome in pediatric obesity
design: The Duke Microbiome Shared Resource (MSR) extracted bacterial DNA from stool sample using a MagAttract PowerSoil DNA EP Kit (Qiagen, 27100-4-EP) that allows for the isolation of samples in a 96 well plate format using a Retsch MM400 plate shaker.  Sample DNA concentration was assessed using a Qubit dsDNA HS assay kit (ThermoFisher, Q32854) and a PerkinElmer Victor plate reader. Bacterial community composition in isolated DNA samples was characterized by amplification of theV4 variable region of the 16S rRNA gene by polymerase chain reaction using the forwardprimer 515 and reverse primer 806 following the Earth Microbiome Project protocol (http://www.earthmicrobiome.org/). These primers (515F and 806R) carry unique barcodes that allow for multiplexed sequencing. Equimolar 16S rRNA PCR products from all samples were quantified and pooled prior to sequencing. Sequencing was performed by the Duke Sequencing and Genomic Technologies shared resource on an Illumina MiSeq instrument configured for 250 base-pair paired-end sequencing runs.