The Development And Applications Of A Novel Library Preparation Method For Highly Accurate Quantification Of Antibody Repertoire

Antibody,the antigen-specific immunoglobulins produced by plasma cells,is the main effector molecules for humoral immune response.The huge diversity of antibody is generated through various mechanisms such as V(D)J gene recombination,junction modification,somatic hypermutation and heavy-light chain pairing,enabling antibody to recognize a variety of foreign antigens,thus providing effective protection for the organism.Antibody repertoire refers to the collection of whole antibodies in an individual or tissue at certain time point.At present,amplification of variable region sequences of B cell receptors by 5’ RACE PCR or multiplex PCR,combined with high-throughput sequencing techniques,is the main method for comprehensively assessing the diversity of antibody repertoire.Therefore,PCR is essential in the preparation of antibody repertoire sequencing libraries.However,incomplete primer extension and template mismatch introduce chimeric sequences during PCR,this will lead to inaccurate quantitative analysis of antibody repertoire characteristics and limits the application of antibody repertoire sequencing for interpreting the key clinical and biological problems.Therefore,how to prepare the accurate antibody repertoire sequencing library is still a difficult problem to be solved urgently.At present,the results of most antibody repertoire studies are based on a small number of samples,using large-scale samples to depict the key features of antibody repertoire would thus be of great value for disease prediction,diagnosis and treatment.In this study,we developed a library preparation method with dual barcodes and dual unique molecular identifiers(UMIs)to solve the above problems,which can remove chimeric sequences from sequencing data and obtain accurate antibody sequences effectively.Next,we obtained 295 antibody repertoire data sets of healthy donor through this method and combined with the collection of 1,857 high-quality repertoire data sets to analyze the critical features of antibody repertoire systematically.The results show that:1.52 core V genes were involved in more than 99%of antibody recombination events in the repertoire;2.The recombination between D and J gene segment was characterized by a distal positional bias;3.The number of public clones between two samples follows a linear model;4.The positive selection dominates in somatic hypermutation(SHM)at RGYW motif.Moreover,we combined this method with single-cell RT-PCR to explore high-affinity p53 antibodies in mice immuned with human p53 protein and obtained two high-affinity antibodies.In summary,this study developed a new library preparation method with dual barcodes and dual unique molecular identifiers(UMIs)for highly accurate antibody repertoire sequencing.With this powerful method,we systematically depicted the critical features of antibody repertoire and constructed a rapid and accurate approach for obtaining two high-affinity p53 antibodies.This library preparation method may be of great value for improving the accuracy and efficiency of antibody repertoire research and provides a new strategy for the development of vaccines and therapeutic antibodies.