Flo min106 r9.4.1 flow cell

The Qiagen Genomic-tip 20/G kit (Qiagen) was used to extract the total genomic DNA following the manufacturer’s recommendations. For Illumina sequencing by MiniSeq, a Nextera XT Library Prep Kit and a Nextera XT Index Kit was used to prepare the DNA library (Illumina, San Diego, CA, United States) according to the manufacturer’s instructions. For Nanopore sequencing by GridION, construction of the library was performed by the SQK-RBK004 Rapid Barcoding Kit (Oxford Nanopore Technologies, Oxford, United Kingdom). The library was loaded onto a FLO-MIN106 R9.4.1 flow cell and sequenced with the GridION device (Oxford Nanopore Technologies, Oxford, United Kingdom). A hybrid assembly of MiniSeq short reads and Nanopore long reads was achieved by Unicycler (Wick et al., 2017 (link)). The annotation was performed using DFAST.³ The complete genome sequences of the two E. coli strains were investigated at the Center for Genomic Epidemiology⁴ using ResFinder-4.1 (identity threshold for gene predictions was 90%), MLST 2.0, pMLST 2.0, VirulenceFinder-2.0 and PlasmidFinder-2. Genomic comparisons were performed using the BRIG tool⁵ and EasyFig tool.⁶ The BLAST program⁷ and ISfinder⁸ were used to analyze the plasmids.

The amplified DNA obtained from Site 57 has also been sequenced by means of long-read Oxford Nanopore Technology platform. It was not possible to apply the same technology to the non-amplified DNA since the minimal amount of required DNA is currently 1 µg. The amplified DNA used for long-read sequencing was not fragmented and the sequencing library was prepared with the standard ligation sequencing kit LSK109 (Oxford Nanopore Technologies, Oxford, UK), adjusting the DNA repair and end-prep incubation step to 20 min. Sequencing was conducted on a MinION equipped with a FLO-MIN106 (R 9.4.1) flowcell (Oxford Nanopore Technologies) for 48 h with the base calling mode set as active.