A Practical Guide to Quantitative Interactor Screening with Next-Generation Sequencing (QIS-Seq)
Yeast two-hybrid screens are a powerful approach to identify protein-protein interactions; however, they are typically limited in the number of interactions identified, and lack quantitative values to ascribe confidence scores to the interactions that are obtained. We have developed a high-throughput, quantitative, yeast two-hybrid screening approach coupled with next-generation sequencing. This strategy allows the identification of interacting proteins that are preferentially associated with a bait of interest, and helps eliminate nonspecific interacting proteins. The method is high-throughput, allowing many more baits to be tested and many more candidate interacting proteins to be identified. Quantitative data allows the interactors to be ascribed confidence scores based on their enrichment with particular baits, and can identify both common and rare interacting proteins.
Key wordsYeast two-hybrid screen Next-generation sequencing High-throughput screening Quantitative
We would like to thank Maël Baudin, Jana A. Hassan, and Vasanth Singan for critically reviewing the manuscript. This work was supported by Natural Sciences and Engineering Research Council of Canada awards to D.S.G. and D.D; a Canada Research Chair in Plant-Microbe Systems Biology (D.D.) or Comparative Genomics (D.S.G.); the Centre for the Analysis of Genome Evolution and Function (D.D. and D.S.G.); United States Department of Agriculture Agricultural Research Service 5335-21000-040-00D (J.D.L).
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