title: Uncultured Microbiome from Ind3
design: DNA isolated from the fecal microbiota was quantified by qPCR, and the V5-V6 regions of the 16S rRNA gene were PCR amplified with forward primer 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGRGGATTAGATACCC-3', and reverse primer 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCGACRRCCATGCANCACCT-3'. Nextera indexing primers were added in the next PCR using the V5F primer 5-AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTC-3, and V6R 5-CAAGCAGAAGACGGCATACGAGAT[i7]GTCTCGTGGGCTCGG-3, where [i5] and [i7] refer to the index sequences used by Illumina. This PCR was carried out using the KAPA HiFidelity Hot Start polymerase (Kapa Biosystems) for 20 cycles. The amplicons were then diluted 1:100 and used as input for a second PCR using different combinations of forward and reverse indexing primers for another 10 cycles. The pooled, size-selected product was diluted to 8pM, spiked with 15% PhiX and loaded onto an Illumina MiSeq instrument to generate the 16S rRNA gene sequences. Samples were heat denatured at 96 degrees C for 2 minutes prior to loading, and a MiSeq 600 cycle v3 kit was used to sequence the sample, resulting in 2.2 million raw reads per sample, on average. Barcodes were removed from the sample reads by UMGC and the Nextera adaptors were trimmed using CutAdapt 1.8.1.