Ampure xp pcr magnetic beads

DNA was isolated in a laminar flow hood to avoid contamination. Genomic DNA was extracted from 1 ml of urine using previously validated protocols^{6 (link)–8 (link)}. Variable region 4 (V4) of the bacterial 16S rRNA gene was amplified via a two-step nested polymerase chain reaction (PCR) protocol, using modified universal primers 515F and 806R, as previously described^{6 (link), 87}. Two quality controls assessed the contribution of extraneous DNA from laboratory reagents: a DNA extraction negative control with no urine added and a PCR negative control with no template DNA added. The final PCR product was purified from unincorporated nucleotides and primers using Qiaquick PCR purification kit (Qiagen, Valencia, CA) and Agencourt AMPure XP-PCR magnetic beads (Beckman coulter). Samples were normalized to equal DNA concentration, as determined by Nanodrop spectroscopy (Thermo Scientific, Waltham, MA). The sample library and the PhiX sequencing control library (Illumina) were denatured and added to the 2×250 bp sequencing reagent cartridge, according to manufacturer’s instructions (Illumina).

Microbial composition was determined by sequencing the variable 4 (V4) region of the bacterial 16S rRNA gene, as described [2 , 6 (link), 9 , 11 ]. The V4 region is ~250 bp, ideal for MiSeq sequence technology (Illumina), and sufficient to classify most bacteria to the family or genus level [15 (link)–18 (link)]. DNA isolation was performed in a laminar flow hood to avoid contamination. Genomic DNA was extracted from 1 ml of urine, using validated protocols [2 , 6 (link), 19 (link)]. The V4 region was amplified by a two-step polymerase chain reaction (PCR), using modified universal primers 515F and 806R, as described [2 , 6 (link)]. Extraction negative controls (no urine) and PCR negative controls (no template) were included to assess contribution of extraneous DNA from reagents. Final PCR products were purified from unincorporated nucleotides and primers using Qiaquick PCR purification kit (Qiagen, Valencia, CA) and Agencourt AMPure XP-PCR magnetic beads (Beckman coulter). Purified samples were normalized to equal DNA concentration, as determined by Nanodrop spectroscopy (Thermo Scientific, Waltham, MA). The sample library and PhiX sequencing control library (Illumina) were denatured and added to the 2x250 bp sequencing reagent cartridge, according to manufacturer’s instructions (Illumina).