Expression modulating variant (EmVar) and allelic effect (AE) variant identification.

Oligo counts were normalized by DESeq2 (¹²) and modeled as a negative binomial distribution to obtain estimates of variance in oligo counts across all samples. EmVars – defined as variants where one or more alleles altered GFP expression (emAlleles) – were determined for the plasmid controls and library replicates and tested for significant expression differences using a Wald’s test (⁶). A fold change (FC) difference of 1.5 between the plasmid controls and EBV library replicates and an FDR<0.05 were required for significance. EmVars were then assessed for allele-specific transactivation potential (allelic effects, AE) by comparing log₂ ratios of the reference versus alternate alleles using a Student’s t-test (⁶). A FC=1.25 between the two alleles and an FDR<0.05 were required for significance. For the 320 multiallelic variants, the reference allele was compared to each alternate allele separately.