Base-resolution stratification of cancer mutations using functional variomics

Abstract

SUMMARY A complete understanding of human cancer variants requires new methods to systematically and efficiently assess the functional effects of genomic mutations at large scale. Here we describe a set of tools to rapidly clone and stratify thousands of cancer mutations at base resolution. This protocol provides a massively parallel pipeline to achieve high stringency and throughput. The approach includes high-throughput generation of mutant clones by Gateway, confirmation of variant identity by barcoding and next-generation sequencing, as well as stratification of cancer variants by multiplexed interaction profiling. Compared with previous site-directed mutagenesis methods, our protocol requires less sequencing effort and enables robust statistical calling of allele-specific effects. To ensure the precision of variant interaction profiling, we further describe two complementary methods, high-throughput enhanced yeast two-hybrid (HT-eY2H) assay and mammalian cell-based Gaussia princeps luciferase protein-fragment complementation assay (GPCA). These independent assays with standard controls validate mutational interaction profiles with high quality. This protocol provides experimentally derived guidelines for classifying candidate cancer alleles emerging from whole-genome or exome sequencing projects as drivers or passengers. For ~100 genomic mutations, the protocol including target primer design, variant library construction and sequence verification can be achieved within as little as 2-3 weeks, and cancer variant stratification can be completed within 2 weeks.

Solution structure of Gaussia Luciferase with five disulfide bonds and identification of a putative coelenterazine binding cavity by heteronuclear NMR

“…Luciferase (Luc) is a generic term for bioluminescent enzymes that catalyze the oxidation of a substrate, often termed luciferin ( 1 , pages xix-xxi). Together with GFP, Luc is widely employed as a reporter protein 2 – 4 . Gaussia Luciferase (GLuc) is a luciferase isolated from the marine Gaussia princeps 5 , which catalyzes a bright blue light by oxidizing coelenterazine.…”

e-MutPath: computational modeling reveals the functional landscape of genetic mutations rewiring interactome networks

“…We implemented a site-directed mutagenesis pipeline to generate specific mutations as in our previous studies ( 5 , 36 ). The mutation clones were sequenced by GENEWIZ and the sequences were confirmed based on the Basic Local Alignment Search Tool programs.…”

“…We first checked whether the sequences were mapped to the exact genes of interest and then verified whether desired base mutations were successfully cloned into the correct position of each gene. We next used the Y2H assay to interrogate mutation-induced PPI alterations based on our previous pipeline ( 5 , 36 ). All protein interaction assays were performed twice independently.…”

Multi-parametric analysis of 57 SYNGAP1 variants reveal impacts on GTPase signaling, localization, and protein stability

“…While many mutations in SYNGAP1 are identified as pathogenic early termination/truncating variants, there is a growing number of missense variants of unknown significance (VUS) for which potential contribution to disease development is unclear. To strengthen clinical relevance of in vitro findings, and allow for structure-function prediction, functional variomics provides multi-dimensional, deep phenotypic characterization of diseaseassociated missense variants [56][57][58][59][60] . Here, we use high-throughput multiplex-phospho flow cytometry and high-content screening to measure the impact of 58 variants on protein localization, stability and function in multiple disease-associated signaling pathways, including pERK, .…”