Assessment of Variant Abundance by Massively Parallel Sequencing for PTEN and TPMT. Assessment of Variant Abundance by Massively Parallel Sequencing for PTEN and TPMT

Determining the pathogenicity of human genetic variants is a critical challenge, and functional assessment is often the only option. Experimentally characterizing millions of possible missense variants in thousands of clinically important genes will likely require generalizable, scalable assays. Here we describe Variant Abundance by Massively Parallel Sequencing (VAMP-seq), which measures the effects of thousands of missense variants of a protein on intracellular abundance in a single experiment. We applied VAMP-seq to quantify the abundance of many thousands of single amino acid variants of two proteins, PTEN and TPMT, in which functional variants are clinically actionable. Overall design: Barcoded single amino acid variant libraries of EGFP-fused PTEN or TPMT were recombined into HEK293T cells previously engineered to contain a Bxb1 recombination site at the AAVS1 locus. Upon sorting cells for EGFP expression, genomic DNA was extracted. Barcodes were amplified and counted with Illumina sequencing. Barcodes were associated back to the corresponding PTEN or TPMT variant by comparison with a barcode-variant map previously created by sequencing the variant library plasmids using PacBio sequencing. Copyright 2018 University of Washington. Data and Scores are owned by the University of Washington. Permission is hereby granted to use, reproduce, and distribute the Data and Scores for noncommercial academic research purposes only, provided that (i) credit for source and copyright are included with each copy and (ii) a link to the original material is provided whenever the material is published elsewhere on the Web. For questions regarding use by non-profit entities or commercial purposes contact: Douglas Fowler, [e-mail]