Whole-Genome Sequencing of Evolved Bacterial Strains

Single-end 36 bp sequencing libraries were created using the Illumina Genomic DNA Sample Prep Kit according to manufacturer's instructions (5 µg input genomic DNA), and sequencing flow cells were prepared using the Illumina Standard Cluster Generation Kit. Samples were sequenced on the Illumina Genome Analyzer II, and image analysis and data extraction were performed using Illumina RTA 1.5.35.0. Reads (with qualities) were aligned to the K12 reference genome (gi|49175990|ref|NC_000913.2) using BWA v0.5.8 [147] (link) with default parameters. Whole-genome pileup files were generated using SAMtools v0.1.8–18 [148] (link) and single-nucleotide polymorphisms due to the evolution were called using custom perl scripts that compared each evolved strain with the original ancestor. Briefly, SNPs passed the filter if they were represented in at least 40% of reads in the evolved strain and at most 10% in the reference strain, with at least 5 reads covering the position in both strains. Additional heuristic filters included a confirming read from both strands, and no more than one ambiguous SNP call (“N”) or deletion (“*”) at that position. Insertions and deletions relative to the K12 reference sequence were identified using Breseq v. 0.18 with default parameters (http://barricklab.org/breseq; [32] (link)).