Uncultured microbiome replicate 2
DNA isolated from the fecal microbiota was quantified by qPCR, and the V5-V6 regions of the 16S rRNA gene were PCR amplified. Nextera indexing primers were added in the first 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 (v3 kit, PE 2 x 300), 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.