title: 16s of homo sapiens: adult gut
design: Sequencing on the 16s rRNA V4 region was performed at the University of Minnesota Genomics Center usng the protocol described in Gohl et. al, 2016. DNA isolated from fecal samples was quantified with qPCR and the V4 region of the 16s rRNA gene was amplified using PCR with barcodes for multiplexing. The qPCR step starts with an initial denaturing step at 95 C for 5 min followed by 35 cycles of denaturation (20s at 98 C), annealing (15s at 66 C) and elongation (1 min at 72 C). After qPCR, samples are normalized to 167,000 molecules/l. This is based on the volume of sample used for PCR1 (3ul), so 500,000 molecules is roughly 10x the target sequencing coverage. The next PCR (PCR1) step is similar to the qPCR step, except with only 25 cycles of denaturation, annealing and elongation. After the first round of amplification, PCR1 products are diluted 1:100 and 5l of 1:100 PCR1 is used in the second PCR reaction. The next step (PCR2) is similar to the previous two PCR protocols, except with only 10 cycles of denaturation, annealing and elongation. Next, Pooled samples were denatured with NaOH, diluted to 8 pM in Illuminas HT1 buffer, spiked with 15% PhiX, and heat denatured at 96 C for 2 minutes immediately prior to loading. A MiSeq 600 cycle v3 kit was used to sequence the sample.