Qubit 4.0 fluorometer assay kit

Whole genome sequencing was performed at the Noguchi Memorial Institute for Medical Research, University of Ghana under the SEQAFRICA Project. Briefly, the DNA for all ESBL-EC isolates from overnight culture were extracted and purified using Qiagen Kit following the manufacturer’s instructions. This was followed by quantification of DNA concentrations using the Qubit 4.0 Fluorometer Assay Kit (Thermo Fisher Scientific, MA, USA). Thereafter, libraries were prepared using the Nextera Flex Kit according to the manufacturer’s instructions. The libraries were quantified using the 2100 Bioanalyzer System (Agilent) and Kapa Sybr Fast qPCR Kit. DNA from each isolate were pooled together and sequenced on an Illumina Miseq platform using a 2 × 300 paired-end approach (Illumina Inc., San Diego, CA, USA). Raw sequencing reads (fastq files) were quality filtered to a Phred score ≥20, filtered for a minimum read length of 50 bp, and adaptor trimmed using Trimmomatic (http://www.usadellab.org/cms/index.php?page=trimmomatic). FastQC tool was used to assess quality of reads (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The resultant high-quality reads were used for de novo assembly using the Unicycler assembler v0.4.9 (Wick et al., 2017 (link)).

The DNA of all the E. coli isolates was extracted using the Lucigen MasterPure™ Gram Positive DNA Purification Kit (ScienceVision, Selangor Darul Ehsan, Malaysia) following the Whole-Genome DNA Isolation for Gram-Negative Bacteria protocol according to the instructions of the manufacturer. This was followed by DNA quantification using the Qubit 4.0 Fluorometer assay kit (Thermo Fisher Scientific, Waltham, MA). Thereafter, libraries for WGS were prepared for each E. coli isolate using a Nextera XT DNA Library Preparation kit (Illumina Inc., San Diego, CA). For each isolate, high-quality Illumina MiSeq (Illumina Inc., San Diego, CA) deep shotgun 2 × 250 bp paired-end reads were assembled de novo into the draft genome sequence using SPAdes assembler v.3.13.1 [30 (link)].