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.