Publications
Nucleic acids researchOct 2025 |
53
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19
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DOI:
10.1093/nar/gkaf1001

A versatile platform for combinatorial antibody library cloning and NGS-based quality control with high accuracy

Zhang, Zhongyao; D'Hondt, Simon; Van der Kant, Rob; Hermans, Guy; Geukens, Nick; Pinheiro, Vitor Bernardes; Schymkowitz, Joost; Rousseau, Frederic; Dewilde, Maarten
Product Used
Genes
Abstract
Synthetic oligo pools offer a cost-effective way for generating hundreds of thousands of antibody sequences but pose technical challenges, particularly in constructing full-length, complex antibody libraries and minimizing chimera formation during polymerase chain reaction (PCR) amplification. In this study, we developed a versatile combinatorial library cloning method capable of constructing single-domain antibody (VHH) and antigen-binding fragment (Fab) libraries with diversities reaching tens of thousands and lengths extending up to four complementarity-determining regions (CDRs). Our protocol allows to largely avoid chimera formation or, if desired, to induce full CDR recombination, starting from the same synthetic oligo pool. This method achieved a full-length rate exceeding 90% for both VHH and Fab libraries and a perfect construct error-free rate above 80% for VHH libraries. We also investigated various PCR conditions and validated that lowering template concentration during amplification effectively reduces chimera formation. Additionally, we established a PCR-free next-generation sequencing (NGS) platform to assess the quality of assembled VHH libraries. Overall, these advancements provide a more efficient approach for constructing high-diversity multi-CDR combinatorial libraries with high coverage and accuracy, along with a reliable NGS preparation method for quality control and downstream analysis, aiding the rapid discovery and development of novel therapeutic biologics.
Product Used
Genes

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