Examining Selection Dynamics and Limitations in Multi-round Protein Selection of High Diversity Libraries

Abstract Proteins and peptides underpin essential biological functions and technological applications, from targeting disease-relevant interactions to providing broad enzymatic activities. However, engineering molecules with desired properties remains difficult, owing to complex sequence-structure-function relationships and the lack of data on specific systems. Experimental selection strategies, including directed evolution, phage display, and mRNA display, address this challenge by leveraging high diversity libraries and iterative enrichment under defined selection pressures. This allows for the identification of candidates without requiring extensive prior knowledge, and can generate extensive datasets for use in machine learning. While many selection systems exist, comparisons across different selection approaches are hindered by the lack of a unifying analytical framework. Here, we present a set of broadly applicable analyses for assessing selection dynamics in multi-round or multi-condition experiments, ranging from position level analysis of sequence properties to full sequence space mappings through protein language model embeddings. Using the toolset to analyze a variety of different datasets in parallel, we explore the potential effects of diversity, coverage, and reproducibility, offering generalizable insights to guide experimental design, interpretation, and troubleshooting across protein and peptide discovery platforms. Competing Interest Statement The authors have declared no competing interest.