Next-generation sequencing (NGS) technologies have been employed in several phage display platforms for analyzing natural and synthetic antibody sequences and for identifying and reconstructing single-chain variable fragments (scFv) and antigen-binding fragments (Fab) not found by conventional ELISA screens. In this work， we developed an NGS-assisted antibody discovery platform by integrating phage-displayed， single-framework， synthetic Fab libraries. Due to limitations in attainable read and amplicon lengths， NGS analysis of Fab libraries and selection outputs is usually restricted to either VH or VL. Since this information alone is not sufficient for high-throughput reconstruction of Fabs， we develo... More
Next-generation sequencing (NGS) technologies have been employed in several phage display platforms for analyzing natural and synthetic antibody sequences and for identifying and reconstructing single-chain variable fragments (scFv) and antigen-binding fragments (Fab) not found by conventional ELISA screens. In this work， we developed an NGS-assisted antibody discovery platform by integrating phage-displayed， single-framework， synthetic Fab libraries. Due to limitations in attainable read and amplicon lengths， NGS analysis of Fab libraries and selection outputs is usually restricted to either VH or VL. Since this information alone is not sufficient for high-throughput reconstruction of Fabs， we developed a rapid and simple method for linking and sequencing all diversified CDRs in phage Fab pools. Our method resulted in a reliable and straightforward platform for converting NGS information into Fab clones. We used our NGS-assisted Fab reconstruction method to recover low-frequency rare clones from phage selection outputs. While previous studies chose rare clones for rescue based on their relative frequencies in sequencing outputs， we chose rare clones for reconstruction from less-frequent CDRH3 lengths. In some cases， reconstructed rare clones (frequency ∼0.1%) showed higher affinity and better specificity than high-frequency top clones identified by Sanger sequencing， highlighting the significance of NGS-based approaches in synthetic antibody discovery.
