Instrument: Illumina MiSeq
Strategy: OTHER
Source: GENOMIC
Selection: other
Layout: SINGLE
Construction protocol: DNA was isolated by Powersoil (QIAGEN, Germantown, MD) per the manufacturer's protocol and recovery yield and DNA quality was determined by fluorometric analysis. DNA concentration was standardized and amplified using 16s rRNA primers spanning the V3 and V4 regions (Illumina). Resulting amplified PCR products were analyzed on a Bioanalyzer (Agilent Technologies, Santa Clara, CA) then purified and amplified with primers containing unique sample nucleotide barcodes (Illumina). PCR products were analyzed with the Bioanalyzer for product quality control and also by SYBR green PCR to determine the quantity. All samples were pooled and standardized to a final concentration of 4.0 nM representation for each sample. The 16S PCR product pool was denatured with sodium hydroxide then adjusted to 4.0 pM and combined with 5% PhiX control DNA prior to loading onto a sequencing flow cell (Illumina) with 300 bp paired ends and a unique molecular tag for each sample.