Extended Data Fig. 2: Validation of SARS-CoV-2 ratio determination by Sanger sequencing.

a–l (Left panels), The correlation between input PFU ratios and output RT-PCR amplicon ratios determined by Sanger sequencing. WT and Mutant viruses were mixed at PFU ratios of 10:1, 5:1, 3:1, 1:1, 1:3, 1:5, or 1:10. Total RNAs of the virus mixtures were extracted and amplified by RT–PCR. The WT/Mutant ratios were calculated by the peak heights of Sanger sequencing. Data were analyzed by linear regression with correlation coefficients (r) and significance (P). Symbols represent individual replicates, and the solid line represents the fitted line. a–l (Right panels), Assay range evaluation. The ratio of wt/Mutant virus mixtures calculated from Sanger sequencing were consistent when using a wide range of virus amounts. The WT/Mutant viruses were mixed at 1:1 PFU ratio. The total titers of the mixed viruses were 10⁶, 10⁵, 10⁴, 10³, and 10² PFU. The total RNA of virus mixture was isolated and amplified by RT-PCR. The wt/Mutant ratios were calculated by the peak heights from Sanger sequencing. Symbols represent individual replicates, bar heights represent the mean, and error bars represent the standard deviation. a–l, Data are derived from a single experiment conducted in triplicate (n = 3).