Whole Genome Sequencing of SARS-CoV-2 using Nanopore

The whole genome of SARS-CoV-2 from the clinical samples was sequenced using the Oxford Nanopore sequencing technology (Oxford Nanopore Technologies, Cambridge, United Kingdom) following the ARTIC network protocol. The SARS-CoV-2 positive RNA extracts were reverse-transcripted with LunaScript^™ RT SuperMix Kit (New England Biolabs, Ipswich, United States). Multiplex PCR with ARTIC Network V3 primer pools, tilling the complete SARS-CoV-2 genome, was performed on cDNA using Q5 Hot Start High-Fidelity DNA polymerase (New England Biolabs, Ipswich, United States). The amplicons were cleaned up with AMPure XP beads (Beckman Coulter Diagnostics, California, United States), and libraries were prepared using the ligation sequencing kit (SQK-LSK109) from Oxford Nanopore Technologies (Oxford, United Kingdom). Then, libraries were quantified using QUBIT 1X dsDNA HS Assay Kit (Invitrogen, Waltham, United States), and 15 ng of each prepared library was loaded into Oxford Nanopore MinION SpotON Flow Cells FLO-MIN106D, R9.4.1 (Oxford Nanopore Technologies, Oxford, United Kingdom (Bull et al., 2020 (link); Pater et al., 2021 (link)). The FastQ files generated by the Mk1C device were used for analysis following the ARTIC Network analysis workflow and EP2ME-lab.