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Short Read-Length Next Generation DNA Sequencing of Antibody CDR Combinations from Phage Selection Outputs

 September 5, 2021



doi: 10.1007/978-1-0716-1450-1_6.

Affiliations

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Landon Pastushok et al.


Methods Mol Biol.


2022.

Abstract

Phage display is commonly used to select target-binding antibody fragments from large libraries containing billions of unique antibody clones. In practice, selection outputs are often highly heterogenous, making it desirable to recover sequence information from the selected pool. Next Generation DNA Sequencing (NGS) enables the acquisition of sufficient sequencing reads to cover the pool diversity, however read-lengths are typically too short to capture paired antibody complementarity-determining regions (CDRs), which is needed to reconstruct target-binding antibody fragments. Here, we describe a simple in vitro protocol to bring the DNA encoding the antibody CDRs closer together. The final PCR product referred to as a “CDR strip” is suitable for short read-length NGS. In this method, phagemid ssDNA is recovered from antibody phage display biopanning and used as a template to create a heteroduplex with deletions between CDRs of interest. The shorter strand in the heteroduplex is preferentially PCR amplified to generate a CDR strip that is sequenced using NGS. We have also included a bioinformatics approach to analyze the CDR strip populations so that single antibody clones can be created from paired CDR sequences.


Keywords:

Antibody; Complementary Determining Region; Next Generation Sequencing; Phage Display.

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