title: 16S data - stool - ICUpatient
design: The V4 hypervariable region of the 16S rRNA gene was amplified using a universal forward sequencing primer and a uniquely barcoded reverse sequencing primer. Primers contained an adapter sequence to bind the amplicons to the Illumina flow cell. PCR-based library construction was performed in triplicate 25 _L solutions containing 1X KAPA2G Robust HotStart ReadyMix, 600 nM each of primer, and 1 _L of DNA template. For every PCR reaction, sterile dH2O was used as a negative control to ensure no contaminating DNA was present. PCR conditions were 95ÁC for 3 min, followed by 31* cycles of 95ÁC for 15 s, 50ÁC for 15 s, 72ÁC for 15 s and were completed at 72ÁC for 5 min. All PCR reactions were run on a 1% agarose TBE gel to visualize the amplification and approximate DNA quantity. Triplicates were pooled together and then combined by approximately even concentrations based on the gel images to create the final library. The final library was purified using 0.8X volumes of Agencourt AMPure XP beads (Beckman Coulter, Indianapolis, IN) according to the manufacturerÍs protocol and quantified using the Qubit Fluorometer. The final library was prepared according to the MiSeq user guide, diluted to a concentration of 7 pM and combined with a 15% PhiX control. Sequencing was performed using the V2 (150bp x 2) chemistry on the Illumina MiSeq platform.