Virus variant determination
VIRUS, MUTATION
Viral genetic variants were identified using real-time PCR genotyping with the TaqMan SARS-CoV-2 Mutation Panel. The variants circulating in Thailand from September 30, 2021 to April 18, 2022 were Delta (B.1.617.2), Omicron BA.1 (B.1.1.529), and Omicron BA.2 subvariants (B.1.1.529). All samples were tested for four canonical mutations of the circulating variants. Those with mutation S.T19R.ACA.AGA were designated Delta (B.1.617.2), if no other mutations in the panel were identified. Those with mutation S.Q493R.CAA.CGA were designated as Omicron BA.1 (B.1.1.529) if no other mutations in the panel were identified. Those with mutations S.Q493R.CA.A.CGA, S.T376A.ACT.GCT, and S.V213G.GTG.GGG were designated Omicron BA.2 (B.1.1.529), if S.T19R.ACA.AGA was absent.Identifications were confirmed using Whole-Genome Sequencing as below:The sequencing method carried out in this experiment follows the ‘PCR tiling of SARS-CoV-2 virus with rapid barcoding and Midnight RT PCR Expansion’ provided by Oxford Nanopore Technology (Oxford, UK) developed based on a protocol by the ARTIC network group. Library preparation process started with reverse transcription, which consists of mixing the purified viral RNA with LunaScript RT SuperMix and incubating the mixtures in a thermal cycler. DNA fragments to be used in the assembly process were amplified by PCR using Midnight primer set (V3) and attached with barcodes from Rapid Barcode Plate (RB96). The mixtures from each sample were pooled together, cleaned with AMPure XP Beads (AXP), and attached with Rapid Adapter F (RAP F). The prepared DNA fragments were then loaded into a primed flow cell (FLO-MIN106) and sequenced on GridION MK1 system.