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| Status |
Public on Mar 13, 2018 |
| Title |
Assessment of Variant Abundance by Massively Parallel Sequencing for PTEN and TPMT |
| Organisms |
Escherichia coli; Homo sapiens |
| Experiment type |
Other
|
| Summary |
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.
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| 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, dfowler@uw.edu
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| Contributor(s) |
Fowler DM, Shendure J |
| Citation(s) |
29785012 |
| NIH grant(s) |
| Grant ID |
Grant title |
Affiliation |
Name |
| R01 GM109110 |
Large-Scale Methods for Assessing the Consequences of Mutations in Proteins |
UNIVERSITY OF WASHINGTON |
Douglas M Fowler |
| R24 GM115277 |
F-CAP: Functionalization of Variants in Clinically Actionable Pharmacogenes |
UNIVERSITY OF WASHINGTON |
Douglas M Fowler |
| DP1 HG007811 |
Interpreting Genetic Variants of Uncertain Significance |
UNIVERSITY OF WASHINGTON |
JAY ASHOK SHENDURE |
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| Submission date |
Jan 03, 2018 |
| Last update date |
Nov 18, 2019 |
| Contact name |
Kenneth Matreyek |
| E-mail(s) |
Kenneth.Matreyek@Case.edu
|
| Organization name |
Case Western Reserve University
|
| Department |
Pathology
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| Lab |
Matreyek
|
| Street address |
2103 Cornell Rd
|
| City |
Cleveland |
| State/province |
OH |
| ZIP/Postal code |
44106 |
| Country |
USA |
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| Platforms (3) |
| GPL18573 |
Illumina NextSeq 500 (Homo sapiens) |
| GPL21222 |
Illumina NextSeq 500 (Escherichia coli) |
| GPL24462 |
PacBio RS II (Escherichia coli) |
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| Samples (20)
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| Relations |
| BioProject |
PRJNA428380 |
| SRA |
SRP127985 |
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