In bulk mRNA samples, 5’ RACE is a popular example. Then a “template switch” primer with poly-G sequence will add adaptor on this side. One-side PCR methods: In the context of using bulk mRNA as template, after reverse transcription, several cytidines are over-hanged to the 3’ end of synthesized cDNA. While when genomic DNA is subject to library preparation, the reverse primers usually anneal to the J intron region. When mRNA (from bulk or single cell) is used as input, reverse primers annealing to C segments or poly-A sequence are commonly used to amplify the V(D)J. Different colors of arrows symbolizes different primer sets targeting various V classes.

Multiplex PCR: regardless of the template types, degenerated primer sets targeting the V gene segments are adopted. In this context, the B cell pools express an advanced antibody repertoire containing highly potent antibodies of different classes. Along with these antigen-dependent processes, naïve mature B cells further differentiate into either antibody-secreting plasma cells or memory B cells B cells expressing mutated variable BCRs undergo affinity-based selection in the secondary lymphoid structure named germinal center, leading to a process named affinity maturation While in SHM, AID can initiate mutation or small indels at IgV exons In CSR, BCR can switch from IgM to another isotype including IgG, IgA, or IgE Upon antigen stimulation, BCR undergoes another layer of diversification, including IgH class-switch recombination (CSR) and IgV somatic hypermutation (SHM), both of which are initiated by activation-induced cytidine deaminase (AID). From the peptide sequence aspect, CDR3 can be identified from several features,Į., starting with a cysteine (C) and ending with phenylalanine (F) or tryptophan (W), as highlighted in the sequence annotation from IMGT (international ImMunoGeneTics information system, As a result, the CDR3 is the most diversified sequence and usually plays the utmost important role during antigen recognition.

The FWRs and CDR1/2 are contributed by the V gene segment, while CDR3 covers a sequence containing the V-D-J junctions. In the IgV domain, the sequence can be further divided into complementarity-determining regions (CDRs) and framework regions (FWRs) The diversity of primary BCR repertoire comes from the numbers of V/D/J gene segments and is also contributed by the insertion and deletion (indel) at the joining junction In this antigen-independent process, the germline variable (V), diversity (D), and joining (J) gene segments are assembled in an ordered manner from a panel of gene segments spanning in the IgV exon to shape a primary antibody repertoire. V(D)J recombination, occurring during B cell development in the omentum, fetal liver, or adult bone marrow, assembles the Ig genes undergo two layers of diversification processes to contribute to the total BCR/antibody repertoire, including the antigen-independent and antigen-dependent processes. To recognize different antigens, in each B cell, Two Layers of BCR Diversification Processes In this review, different BCR repertoire library preparation protocols are outlined and three major steps of BCR-seq data analysis,Į., V(D)J sequence annotation, clonal phylogenetic inference, and BCR repertoire profiling and mining are summarized. The IgV contains highly diversified sequences and is primarily responsible for antigen recognition, while the sequence constant domain can activate downstream immune responses. Both IgH and IgL chains can be divided into the variable N-terminal Ig domain (IgV) and constant C-terminal Ig domain (IgHC or IgLC). Igλ are located at 2p11.2 and 22q11.2, respectively The secreted form of BCR is also called antibody, which contains two identical immunoglobulin (Ig) heavy (IgH) chains and two identical Ig light (IgL) chains The antigen receptor on B cells, B cell receptor (BCR), recognizes the antigen and plays key roles in B cell development, survival and activation.

Antibody repertoire molecular targeting