Amplification and Sequencing of SARS-CoV-2 S Gene

The full-length S gene (approximately 3.9 kb) was amplified by PCR using the cDNA of each TCoV isolate with the primers Sup and Sdown3 (Online Resource 1). The mixture (64:1, v:v) of Taq (Promega Corp., Madison, WI, USA) and Pfu DNA polymerases (Stratagene, La Jolla, CA, USA) with proofreading ability was used in a 96-well thermal cycler (GeneAmp, Perkin-Elmer Cetus Corp., Norwalk, CT, USA) to maintain the fidelity of the PCR [9 (link)]. The PCR products were electrophoresed on 1 % agarose gels and purified using a Zymoclean^TM Gel DNA Recovery Kit (Zymo, Irvine, CA, USA) for further sequencing. Several primers (Online Resource 1) designed to sequence overlapping fragments covering the full length of the S gene of TCoV were used to determine the nucleotide sequences of the purified PCR products at the Purdue University Genomics Core Facility (West Lafayette, IN, USA). In addition, the purified PCR product was cloned into the PCR-II plasmid vector and used to transform E. coli strain TOP10F’ according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA).