Genome Sequencing and Mutation Analysis of E. coli

DNA was isolated from overnight cultures using the Gentra Puregene Yeast/Bacteria kit (Qiagen). Twenty-one multiplex libraries were made using the IntegenX Apollo 324™ System. The libraries were sequenced on a single lane of an Illumina HiSeq, to a read length of 140 bases, resulting in an average fold-coverage of 400 for each strain. The reads were mapped to the E. coli K-12 MG1655 reference genome (GenBank: U00096.2) [22 (link)] using BWA for Illumina [23 (link)]. The resulting SAM files were converted to BAM files using SAMtools [24 (link)]. The BAM files were visualized using Integrated Genome Viewer (IGV) [25 (link)], manually searching the genomes for SNPs, deletions, insertions, and amplifications. An insertion is predicted at a genomic location with a marked decrease in read depth, which occurs when individual sequencing reads fail to span that location. An amplification is predicted at genomic intervals containing a consistent read depth of more than 1.5-times the background read depth. All mutations are described in Additional file 1: Table S1. To reduce the possibility of overlooking some genomic mutations, the sequence data were also analyzed with FreeBayes, a powerful variant-detector package [26 ]. No additional mutations were identified using this second method.