Figure 1.

A) Overview of MAVEs. A library of variants of the genetic element of interest is created and introduced into cells. The cells are subjected to a growth- or fluorescent reporter-based assay. High-throughput sequencing is used to determine the frequency of variants before and after the assay, and variant frequencies are used to calculate functional scores. B) VAMP-seq uses a GFP fusion reporter to measure steady-state variant abundance. GFP was fused N-terminally to a library of TPMT variants; mCherry was used as a transcriptional control. This library was introduced into HEK293T cells using a serine integrase landing pad system such that only one variant is expressed per cell. Cells were sorted based on their fluorescence into four bins. High-throughput sequencing was used to determine the frequency of every variant in each bin. Frequencies were then converted to abundance scores. C) A FACS plot of WT TPMT (red) and three high-frequency variants known to be low abundance (blue): A80P, A154T, and Y240C. The library of TPMT variants and bins used for sorting are shown (gray). D) Density plot of abundance scores, with dotted blue line showing distribution of nonsense variants and red dotted line showing synonymous variants. The missense variant distribution is shaded from blue (low abundance) to red (high abundance). E) Heatmap of TPMT abundance scores shaded from blue (low abundance) to red (high abundance); gray indicates missing data. F) Abundance scores from four replicates for six new TPMT variants found in gnomAD.