Plasmid DNA Isolation and Barcode Amplification

We isolated plasmid DNA from each bulk competition time point as described (Jiang et al., 2013 (link)). Purified plasmid was linearized with AscI. Barcodes were amplified by 19 cycles of PCR using Phusion polymerase (NEB) and primers that add Illumina adapter sequences and an 8 bp identifier sequence used to distinguish libraries and time points. The identifier sequence was located at positions 91–98 relative to the Illumina primer and the barcode was located at positions 1–18. PCR products were purified two times over silica columns (Zymo Research) and quantified using the KAPA SYBR FAST qPCR Master Mix (Kapa Biosystems) on a Bio-Rad CFX machine. Samples were pooled and sequenced on an Illumina NextSeq instrument in single-end 100 bp mode.