scholarly journals Multiplex Assessment of Protein Variant Abundance by Massively Parallel Sequencing

2018 ◽  
Author(s):  
Kenneth A. Matreyek ◽  
Lea M. Starita ◽  
Jason J. Stephany ◽  
Beth Martin ◽  
Melissa A. Chiasson ◽  
...  

ABSTRACTDetermining 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 apply VAMP-seq to quantify the abundance of 7,595 single amino acid variants of two proteins, PTEN and TPMT, in which functional variants are clinically actionable. We identify 1,079 PTEN and 805 TPMT single amino acid variants that result in low protein abundance, and may be pathogenic or alter drug metabolism, respectively. We observe selection for low-abundance PTEN variants in cancer, and our abundance data suggest that a PTEN variant accounting for ~10% of PTEN missense variants in melanomas functions via a dominant negative mechanism. Finally, we demonstrate that VAMP-seq can be applied to other genes, highlighting its potential as a generalizable assay for characterizing missense variants.

2018 ◽  
Vol 50 (6) ◽  
pp. 874-882 ◽  
Author(s):  
Kenneth A. Matreyek ◽  
Lea M. Starita ◽  
Jason J. Stephany ◽  
Beth Martin ◽  
Melissa A. Chiasson ◽  
...  

PLoS Genetics ◽  
2010 ◽  
Vol 6 (8) ◽  
pp. e1001079 ◽  
Author(s):  
Cord Drögemüller ◽  
Jens Tetens ◽  
Snaevar Sigurdsson ◽  
Arcangelo Gentile ◽  
Stefania Testoni ◽  
...  

Author(s):  
Cordula Haas ◽  
Jacqueline Neubauer ◽  
Andrea Patrizia Salzmann ◽  
Erin Hanson ◽  
Jack Ballantyne

Author(s):  
Stella C. Yuan ◽  
Eric Malekos ◽  
Melissa T. R. Hawkins

AbstractThe use of museum specimens held in natural history repositories for population and conservation genetic research is increasing in tandem with the use of massively parallel sequencing technologies. Short Tandem Repeats (STRs), or microsatellite loci, are commonly used genetic markers in wildlife and population genetic studies. However, they traditionally suffered from a host of issues including length homoplasy, high costs, low throughput, and difficulties in reproducibility across laboratories. Massively parallel sequencing technologies can address these problems, but the incorporation of museum specimen derived DNA suffers from significant fragmentation and exogenous DNA contamination. Combatting these issues requires extra measures of stringency in the lab and during data analysis, yet there have not been any high-throughput sequencing studies evaluating microsatellite allelic dropout from museum specimen extracted DNA. In this study, we evaluate genotyping errors derived from mammalian museum skin DNA extracts for previously characterized microsatellites across PCR replicates utilizing high-throughput sequencing. We found it useful to classify samples based on DNA concentration, which determined the rate by which genotypes were accurately recovered. Longer microsatellites performed worse in all museum specimens. Allelic dropout rates across loci were dependent on sample quantity, with high concentration museum specimens performing as well and recovering quality metrics nearly as high as the frozen tissue sample. Based on our results, we provide a set of best practices for quality assurance and incorporation of reliable genotypes from museum specimens.


BioTechniques ◽  
2013 ◽  
Vol 55 (2) ◽  
Author(s):  
Tú Nguyen-Dumont ◽  
Bernard J. Pope ◽  
Fleur Hammet ◽  
Melissa C. Southey ◽  
Daniel J. Park

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