title: Rectal swab isolate 30AD4R
design: Automated amplicon library preparation was performed on a Biomek 4000 using a custom liquid handling protocol. The V4 region of the 16S rDNA gene was amplified using barcoded primers.  58 Each unique barcoded amplicon was generated in pairs of 25 L reactions with the following reaction conditions: 8.5 L PCR-grade H2O, 12.5 L GoTaq G2 Master Mix (Cat. No. M7822, Promega, Wisconsin), 2 L each of barcoded forward and universal reverse primers (5 M) and 2 L 100x diluted template DNA. Reactions were run on a C1000 Touch Thermal Cycler (Bio-Rad, California) with the following cycling conditions: initial denaturing at 94C for 3 min followed by 35 cycles of denaturation at 94C for 45 sec, annealing at 50C for 1 min, and extension at 72 C for 90 sec, with a final extension of 10 min at 72C. Amplicons were quantified using the 2200 TapeStation system (Agilent, California) and Quant-iT 1x dsDNA HS Assay Kit (Cat. No. Q3323, Invitrogen, Massachusetts) and pooled equimolar. Purification of the pool was then performed using 0.8x AMPureXP beads (Cat. No. A63880, Beckman Coulter, California) as per the manufacturer instructions and final concentration determination was determined using Qubit (Invitrogen, Massachusetts). Sequencing was performed using paired-end 150 cycles on a MiSeq (Illumina) using custom spike-in Read 1/Index/Read 2 primers.