Megabace 500

Total mRNA from parental and CDV^R cells was isolated with the Quickprep mRNA purification kit (Amersham Biosciences) and converted to cDNA with the first-strand cDNA synthesis kit (GE Healthcare). The entire cDNA from each selected gene was amplified by PCR using specific primers. The PCR products were purified using PCR product purification kit (Roche) and directly sequenced using a cycle-sequencing kit (Dyenamic dye terminator kit; Amersham Biosciences), specific primers targeting both strands of the specific gene, and a capillary DNA sequencing system (MegaBACE 500; Amersham Biosciences). The data were assembled and compared to the DNA sequences obtained from reference sequences using Vector NTI software (Invitrogen). The primers used for the genotyping of UMP/CMPK1 and 2, and HPV oncogenes E6 and E7 are listed in Table S2.

Phylogenetic groups were determined by the quadruplex PCR method [22 (link)]. Multilocus sequence typing was carried out by amplifying and sequencing seven conserved housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA), as recommended by the EnteroBase Database (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli). PCR products were sequenced in a MegaBACE 500 sequencer (Amersham Biosciences, Piscataway, NJ, USA). Raw sequences were visually reviewed and edited using the Chromas Lite 2.1 software (Technelysium, South Brisbane, Australia) and aligned with the ClustalW algorithm of the MEGA7 software [23 (link)]. Each gene locus was assigned an allele number and a sequence type (ST) was determined for each isolate according to the allele profile. Grouping of isolates into clonal complexes was done with the eBurst algorithm implemented in the EnteroBase platform.