Sequencing and bioinformatics analysis
Libraries were prepared using the Oxford Nanopore Technologies Midnight Protocol, following the manufacturer’s instructions. Briefly, cDNA was generated from the extracted viral RNA using the NEB LunaScript RT mastermix, then split into two pools for PCR with the midnight primers, generating 1200bp amplicons8,9. Amplicons were pooled and then cleaned up using AmpureXP magnetic beads (Beckman Coulter, USA) before being quantified by fluorimetry using the Qubit 1X HS dsDNA quantification kit and a Qubit 4 platform (Thermo Fisher Scientific, USA). Samples were then barcoded using the rapid barcoding kit (SQK-RBK110.96) following the protocol instructions before another round of AmpureXP cleanup and quantification. Samples were normalized, then adaptor-ligated ready for loading into R9.4.1 nanopore flowcell. Basecalling and demultiplexing were performed by Guppy (Version 5.1.11), integrated with MinKNOW (Version 21.05.8), and the runs allowed to continue for 48 hours to collect as many reads as possible. Consensus was generated using the in-house workflow with a minimum coverage of 30x on Qiagen CLC workbench, and lineages assigned using the Pangolin software.