Geneelute bacterial kit

Whole genome sequencing was performed by SeqCenter, LLC in Pittsburg, PA, USA. Genomic DNA extractions were performed using Sigma GeneElute Bacterial kit. The libraries were prepared using the Illumina DNA Prep kit with IDT 10bp UDI indices and sequenced on an Illumina NextSeq 2000 with 2×151bp reads. Demultiplexing, quality control and adapter trimming was initially performed using Illumina’s bcl-convert v. 3.9.3. TrimGalore! v. 0.6 was additionally used to remove remaining adapters and low-quality reads. Breseq^{67 (link)} v. 0.36.0 was used to identify mutations between ancestral and evolved strains.

To test if upf1 encodes a methyltransferase as computationally predicted we utilized the base modification analysis provided by the Pacbio Sequel II DNA sequencer. The methylase-free E. coli strain ER2796^{43 (link)} with expression vector pCW-LIC-upf31 was tested against ER2796 (pCW-LIC-sacB) for differential base modification^{36 (link)} on a Pacbio Sequel II at the Arizona Genomic Institute in Tucson, AZ, USA. Both strains were grown with antibiotic selection and IPTG induction before genomic extraction using the Sigma GeneElute Bacterial kit. Base Modification Analysis was run on SMRT Link v. 10.2.0.133434 with a reference made by merging the E. coli assembly with the respective plasmids and enabling “Find Modified Base Motifs” and “Consolidate Mapped BAMs for IGV” options. All the other options were left as default.