Extended Data Figure 2. Validation of competition assay by Sanger sequencing.

(a) The correlation between input PFU ratios and output RT-PCR amplicon ratios determined by Sanger sequencing. D614 and G614 viruses were mixed at PFU ratios of 10:1, 5:1, 3:1, 1:1, 1:3, 1:5, or 1:10. Total RNA of the virus mixtures were extracted and amplified by RT-PCR. The D614/G614 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. Data is derived from a single experiment conducted in duplicate. (b) Assay range evaluation. The ratio of D614/G614virus mixture calculated from Sanger sequencing was consistent when using a wide range of virus amounts. The D614/G614 viruses were mixed at 1:1PFU ratio. The total titers of the mixed viruses were 10², 10³, 10⁴, 10⁵, and 10⁶ PFU. The total RNA of virus mixture was isolated and amplified by RT-PCR. The D614/G614 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. Data is derived from a single experiment conducted in triplicate.