Whole genome sequencing of SARS-CoV-2: rdv_paired
Viral RNA was extracted from nasopharyngeal specimens stored in viral transport media (VTM) utilizing the QIAamp 96 Virus QIAcube HT Kit (Qiagen). cDNA synthesis was performed using the SuperScript IV First Strand Synthesis Kit (Thermo) with random hexamer primers, as per the manufacturer's instructions. The viral genome cDNA was then amplified in multiplexed PCR reactions to produce approximately 400 base pair amplicons, covering the entire genome. The multiplex primer set, consisting of two non-overlapping primer pools, was designed using Primal Scheme and provided by the Artic Network (v4 and 4.1). The sequencing libraries were prepared from these amplicon pools using the SeqWell plexWell 384 kit (Seqwell PW384A) according to the manufacturer's protocol. These pooled libraries were sequenced on an Illumina MiSeq platform using the V2 500 cycle kit.