ArticleNucleic acids research2025
A versatile platform for combinatorial antibody library cloning and NGS-based quality control with high accuracy.
Article in Nucleic acids research, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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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.
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