Y2H-SCORES V2 identifies protein-protein interaction domains in yeast two-hybrid next-generation interaction screen data

Authors: VELÁSQUEZ-ZAPATA, VALERIA - Iowa State University; SMITH, SCHUYLER - Oak Ridge Institute For Science And Education (ORISE); Fuerst, Gregory; Wise, Roger

Submitted to: Methods in Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/24/2026
Publication Date: N/A
Citation: N/A

Interpretive Summary: Yeast two-hybrid (Y2H) is a technique to study how proteins interact with each other. This knowledge is required for all functions in the cell, including creating disease resistance crops for U.S. agriculture. To more efficiently discover these interactions, researchers have combined Y2H with advanced sequencing technology, creating a method called next-generation interaction screening (NGIS). A key part of this process involves new, and often, custom software to analyze the big data. One of these softwares, designated Y2H-SCORES, provides researchers detailed information about novel protein-protein interactions. However, to keep Y2H-SCORES up-to-date and working dependably, we have used new technologies called containerization which assembles all the components in one place, simplifying installation and analysis. This updated version is made available in both Singularity and Docker formats, ensuring it works smoothly and consistently for researchers. Impact: This containerization guarantees consistent execution, simplifies installation, and supports long term sustainability of the analytical framework. Together, these developments position Y2H SCORES V2 as a mature, scalable, and domain resolving platform for discovery of protein-protein interactions, and thus, more efficient processing of big data in agriculture to provide a more safe and affordable food supply for American consumers.

Technical Abstract: Y2H-SCORES V2 is an enhanced analytical framework for prioritizing protein–protein interaction candidates in Yeast Two-Hybrid Next-Generation Interaction Screening (Y2H-NGIS) experiments. This updated version extends the original scoring methodology with algorithms specifically optimized to detect interaction-enriched prey fragments, enabling high-resolution identification of binding domains. Y2H-SCORES V2 integrates improved processing of NGPINT V3 outputs including transcript-level abundance estimates and fusion-read counts to compute refined enrichment, specificity, and in-frame scores. To ensure reproducibility and seamless deployment across high performance computing (HPC) and cloud environments, the entire workflow and all dependencies are now fully containerized using Docker and Singularity. This unified, portable pipeline enables robust, scalable, and domain-aware prioritization of candidate interactors for high-throughput Y2H-NGIS studies.