Rapid barcoding sequencing

Whole-genomic sequencing was performed using the Nanopore Rapid Barcoding Sequencing (SQK-RBK004) protocol on a GridlON X5 (Oxford Nanopore, Oxford, UK) sequencing instrument as previously described [10 ]. Briefly, cDNA was generated from the previously extracted RNA remaining after the initial diagnostic RT-PCR assay. The hexamer cDNA was randomly synthesized using SuperScript IV (Thermo Fisher, Waltham, MA, USA, 18091) for two-step reverse transcriptase PCR. For Oxford Nanopore sequencing, amplicon pools were indexed using the Rapid Sequencing Adapter (Oxford Nanopore, Oxford, UK, EXP-RAP001). Then, about 400 ng of the resulting library was used for sequencing on Oxford Nanopore GridION instruments using R9.4.1 flow cells. Clade determination was performed via NextClade CLI v2.14.1 [11 (link)].

Libraries were prepared using the Rapid Barcoding Sequencing or Nanopore Genomic DNA Ligation kit with Native barcoding according to manufacturer’s instructions (Oxford Nanopore Technologies). Libraries were loaded into the R9.4.1 flow cell and run on the MinION MK1B device. After flow cell quality checks, all sequencing utilized the flow cell priming kit (EXP-FLP002) and sequencing commenced at −180 mV; voltage drift was accounted for where the flow cell went through a wash protocol. The sequencing runs were monitored with MinKNOW version 21.05.25 core 4.3.12. Fast basecalling model was applied with Bream version 6.2.6 and Guppy version 5.0.16 (version 3.2.9 used prior to February 2021). Libraries were run for 72 h but produced adequate data for further analysis within 6–12 h in the majority of samples. Final run QC analysis was undertaken with PycoQC prior to bioinformatic analysis.
In addition, pure colonies of bacteria isolated from positive blood cultures were also sequenced, using DNA extracted by QIAGEN DNeasy Ultraclean Microbial Kit, with quantification by Life Technologies QUBIT fluorometer and library preparation with Illumina ILMN DNA LP tagmentation. Library size was determined by Agilent TapeStation 4150 using D1000 High Sensitivity kit. Sequencing was performed on the Illumina MiniSeq platform with the 300 cycle output reagent kit (Fig. 1).