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.
© The Author(s) 2025. Published by Oxford University Press.