Miseq nextera xt dna library preparation kit

Both C. jejuni strains were grown overnight at 42°C on OMHA + blood plates under microaerobic conditions and genomic DNA was extracted using Epicentre Metagenomic DNA Isolation kits for Water (Illumina) according to the manufacturer’s instructions, as previously described (Clark et al., 2016 (link)). Briefly, quantification of DNA was performed by DeNovix QFX Fluorometer using Qubit dsDNA BR assay kit (Fisher Scientific). Sample libraries were prepared using a MiSeq Nextera^® XT DNA library preparation kit (Illumina). Whole genome sequencing was performed by 250 bp paired end read sequencing on the Illumina MiSeq sequencer using a MiSeq^® Reagent Kit V2 and 500 cycles on the Illumina MiSeq platform. Sequence reads were assembled into contigs using INNUca 2.6. Pangenomic annotations were performed by Roary (Supplementary Data S1). Raw reads can be accessed on NCBI under the reference number PRJNA903792. From pangenomic annotations, strain specificity, using the NCBI BLAST tool and conventional PCRs, was verified for many genes. Among these genes, two genes unique to G2008b (LpsA and DmsB) and two genes unique to D2008b (McrBC and RimP) were selected as candidates for qPCR/PCR targets for the quantification/identification of each strain in the samples (Supplementary Figure S1).

Genomic DNA was prepared from isolates cultured overnight at 42°C on OMHA + blood using Epicentre Metagenomic DNA Isolation kits for Water (Illumina) according to the manufacturer’s instructions. DNA was quantitated using Qubit dsDNA BR assay kits (Life Technologies, Invitrogen). Sample libraries were prepared using a MiSeq Nextera^® XT DNA library preparation kit (Illumina). Whole genome sequencing was performed by 250 bp paired-end read sequencing on the Illumina MiSeq sequencer using the MiSeq^® Reagent Kit V2 and 500 cycles on the Illumina MiSeq platform to obtain an average genome coverage of 30–50×. Sequence reads were assembled into contigs using the SPAdes assembler (v3.0) [35 (link)]. Contigs smaller than 1kb and with average genome coverage less than 15× were filtered and removed from the analysis. The remaining contigs were closed and finished using Staden gap v4.10 by read mapping to the reference genome, NCTC11168, plus a combination of PCR and Sanger sequencing for gap closure. Fasta files for each genome were sent to Genomes (NCBI) for annotation using the NCBI prokaryotic annotation pipeline.

Genomic DNA was prepared from four selected Walkerton outbreak isolates cultured overnight at 42 °C on OMHA + blood using Epicentre Metagenomic DNA Isolation kits for Water (Illumina) according to the manufacturer’s instructions. Quantitation of DNA was accomplished using Qubit dsDNA BR assay kits (Life Technologies, Invitrogen). Sample libraries were prepared using MiSeq Nextera® XT DNA library preparation kit (Illumina). Whole genome sequencing was performed by 250 bp paired-end read sequencing on the Illumina MiSeq sequencer using MiSeq® Reagent Kit V2 and 500 cycles on the Illumina MiSeq platform to obtain an average genome coverage of 30–50×. Sequence reads were assembled into contigs using the SPAdes assembler (v3.0 [46 (link)]). Contigs smaller than 1-kb and with average genome coverage less than 15× were filtered and removed from the analysis. The remaining contigs were closed and finished using Staden gap v4.10 by read mapping to the reference genome, NCTC11168, and a combination of PCR and Sanger sequencing for gap closure. Fasta files for each genome were sent to Genomes (NCBI) for annotation using the NCBI prokaryotic annotation pipeline. Additional information for selected loci was then added manually.