VEEVTC83_BDGR49_3200_R1
To sequence virus genomes from cell culture, 0.5 to 1ml of supernatant from each passage was harvested and twice-clarified by centrifugation, and RNA was isolated using TRIzol LS reagent (Invitrogen). cDNA was synthesized from purified RNA using random hexamer primers and Superscript IV. cDNA was amplified through 7 or 30 rounds of PCR, using the Phusion high-fidelity kit and a set of 25 primer pairs, using a tiling approach that doubly covered the whole genome, making PCR fragments either 500bp or 1kb in length. PCR products were purified using the Wizard SV gel and PCR clean-up system (Promega). Libraries for NGS were prepared using the Nextera XT kit (Illumina) and then sequenced on either the Illumina MiSeq or NextSeq 500. Data were analyzed using CLC Genomics Workbench v.12 (Qiagen). To process the sequencing data, reads less than 50 nucleotides in length and with a Phred scoreof <20 were removed. The remaining reads were mapped to the seed stock consensus sequence, and variants were identified either with a minimum frequency above 1% in all reads at positions with a minimum coverage of 400 or, if the quality and quantity of reads were sufficient, with a minimum frequency above 0.5% in all reads at positions with minimum coverage of 1,000