Genomic Sequencing of E. coli TolC

Genomic DNA of E. coli TolC was extracted by gravity flow using the Genomic-tip 20/G kit (Qiagen, Germany). Purified genomic DNA of E. coli TolC was processed for PacBio SMRT sequencing and Illumina MiSeq paired-end sequencing (2 × 250 bp) with a target genome coverage of 150-fold. DNA libraries for MiSeq sequencing of the genome of E. coli TolC were prepared with the NEBNext Ultra DNA library prep kit for Illumina sequencing (New England Biolabs, Ipswich, MA). Quality controls of NEBNext Ultra DNA libraries were conducted by fluorometric quantitation using the Qubit 3.0 fluorometer (Thermo, Fisher Scientific, Germany). For PacBio SMRT sequencing, a PacBio SMRTbell library was constructed according to the manufacturer’s instructions and the library was sequenced on the PacBio RSII platform. De novo genome assemblies were built with PacBio’s SMRT Portal (v.2.3.0) by utilizing the Hierarchical Genome Assembly Process 3 (HGAP3) (46 (link)). The genome was error corrected against indel errors by a mapping of Illumina reads onto finished genomes, using BWA (47 (link)) with subsequent variant and consensus calling using VarScan (48 (link)); automated sequence annotation was performed with Prokka (v.1.8) (49 (link)).