Abstract
Phage display is a powerful technology that has demonstrated great potential in identifying peptides with high binding affinity and specificity toward a broad spectrum of biological targets. The undesired enrichment of nonspecific binders remains a major challenge in phage display selection. The integration of next-generation sequencing (NGS) into phage display has expanded the horizons of ligand discovery by significantly enhancing our ability to interrogate the massive sequence space of combinatorial phage display libraries and providing quantitative information about their composition and evolution during biopanning. NGS findings have provided strong support for the notion that the selection output still contains a large number of nonspecifically enriched peptide sequences that could not be removed or identified by traditional strategies for biopanning optimization. Despite its great potential for increasing the strength of peptide discovery, the routine NGS-based phage display workflow, which relies on analyzing the biopanning output, fails to effectively distinguish thousands of nonspecific peptides from specific target-binding sequences. By incorporating precise control experiments-including the NGS characterization of the unamplified and amplified naïve libraries and the outputs of targetless and replicate selections-alongside the thoughtful data analysis and interpretation, we propose an optimized workflow of NGS-based phage display that would be capable of distinguishing many target-specific peptides from the overwhelming background of nonspecific binders. Applying such a systematic approach will not only advance fundamental research for peptide discovery but also hold promise for the clinic, where these peptides can serve as the foundation for next-generation diagnostic and therapeutic platforms in precision medicine.