RESUMO
Antibody phage-display technology identifies antibody-antigen interactions through multiple panning rounds, but traditional screening gives no information on enrichment or diversity throughout the process. This results in the loss of valuable binders. Next Generation Sequencing can overcome this problem. We introduce a high accuracy long-read sequencing method based on the recent Oxford Nanopore Technologies (ONT) Q20 + chemistry in combination with dual unique molecular identifiers (UMIs) and an optimized bioinformatic analysis pipeline to monitor the selections. We identified binders from two single-domain antibody libraries selected against a model protein. Traditional colony-picking was compared with our ONT-UMI method. ONT-UMI enabled monitoring of diversity and enrichment before and after each selection round. By combining phage antibody selections with ONT-UMIs, deep mining of output selections is possible. The approach provides an alternative to traditional screening, enabling diversity quantification after each selection round and rare binder recovery, even when the dominating binder was > 99% abundant. Moreover, it can give insights on binding motifs for further affinity maturation and specificity optimizations. Our results demonstrate a platform for future data guided selection strategies.