title: UA13_2_T
design: Full-length 16S rRNA gene was amplified using 1-2 g of DNA as template, the universal 16S rRNA bacterial primers 27F and 1492R (0.2 M each), and 1X Hot-Start GoTaq DNA Polymerase Master Mix (Promega) in 50 l final volume. Cycling conditions were 94 C, 3 min; then 35 cycles of 94 C 30 s, 54 C 30 s, 72 C 2 min, and a final extension step at 72 C for 5 min. Barcodes were added to PCR amplicons through a second PCR reaction using Barcoded Universal F/R Primers (Pacific Biosciences, 100-466-100). PCR products were cleaned with AxyPrep MagPCR (Axygen) according to the manufacturers protocol and eluted in 50 ?l of water. Cleaned barcoded 16S rRNA amplicons were quantified using AccuClear Ultra High Sensitivity dsDNA Quantitation Kit (Biotium) on BioTek FLx800 Microplate Fluorescence Reader. Based on quantification results, barcoded amplicons were then pooled in equimolar concentration into multiplexed sets of 2 to 18 samples per pool.