SARS-CoV-2 Genome Sequencing Protocol

Viral RNA was extracted from nasopharyngeal specimens utilizing the QIAamp Viral RNA Minikit (Qiagen). Confirmatory testing for SARS-CoV-2 was performed by qRT-PCR with the CDC 2019-nCoV RT-PCR Diagnostic Panel utilizing N1 and RNase P probes as previously described [IDT, cat. no. 10006713]. RNA samples of sufficient quality (RNaseP control, Ct value <35) and with sufficient copies of the viral genome for sequencing (N1, Ct value <32) were reverse transcribed into complementary DNA (cDNA) [16 (link)]. cDNA synthesis was performed with the SuperScript IV First Strand Synthesis Kit (Thermo) using random hexamer primers according to the manufacturer’s specifications. Direct amplification of the viral genome cDNA was performed in multiplexed PCR reactions to generate ~400 base pair amplicons tiled across the genome. The multiplex primer set, comprised of two non-overlapping primer pools, was created using Primal Scheme and provided by the Artic Network (version 3 release). PCR amplification was carried out using Q5 Hot Start HF Taq Polymerase (NEB) with 5μl of cDNA in a 25μl reaction volume. A two-step PCR program was used with an initial step of 98°C for 30 seconds, then 35 cycles of 98°C for 15 seconds followed by five minutes at 65°C. Separate reactions were carried out for each primer pool and validated by agarose gel electrophoresis.