Assessing the reliability of spike-in normalization for analyses of single-cell RNA sequencing data

Variance estimates of the log₂-ratio between the ERCC and SIRV total counts across wells (A) or the log₂-size factors computed from those totals (B). For the separate/premixed experiments, each estimate is the residual variance of a linear model fitted to the log-ratios across the corresponding wells on each plate. The variance of volume addition is in our framework. For the log-size factors, each estimate is the residual variance of a linear model fitted to all cells on each plate. Results are shown for experiments with 416B cells or TSCs, with two replicate plates for each cell type. Error bars represent the standard errors of the estimates, assuming log-values are normally distributed. Numbers represent the residual degrees of freedom used for each estimate—for B, this was the same for each spike-in set. Differences between the separate-addition and premixed estimates for each batch were assessed using a one-sided F-test, yielding P-values of 0.28, 1.00, 1.00, and 0.06 from left to right.