What is BCR Sequencing
B cell receptor sequencing profiles the antibody repertoire. It reveals which antibodies a person is making, how diverse their immune response is, and how antibodies evolve through somatic hypermutation.
What BCR sequencing is
Section titled “What BCR sequencing is”BCR sequencing reads the immunoglobulin genes from B cells. Every B cell produces a unique antibody. By sequencing these genes across thousands or millions of B cells, you build a catalog of the antibodies present in a sample.
This catalog is called the antibody repertoire. It is a snapshot of the adaptive immune system at a specific moment. The repertoire changes over time in response to infection, vaccination and disease.
BCR sequencing is also called:
- Antibody repertoire sequencing
- Ig-seq (immunoglobulin sequencing)
- Rep-seq (repertoire sequencing)
Why it matters
Section titled “Why it matters”Antibodies are the adaptive immune system’s main weapon against pathogens. Each antibody binds a specific target. The diversity and composition of the antibody repertoire determines how well the immune system can respond to threats.
Most of this diversity is invisible to traditional assays. ELISA measures total antibody levels. Flow cytometry counts B cell populations. Neither reveals the sequence-level diversity of the repertoire.
BCR sequencing fills this gap. It provides single-sequence resolution of the entire antibody pool in a sample.
Antibody structure
Section titled “Antibody structure”An antibody is made of two identical heavy chains and two identical light chains. Each chain has a variable region and a constant region.
The variable region determines what the antibody binds. It contains three hypervariable loops called complementarity-determining regions (CDR1, CDR2, CDR3). Among these, CDR3 of the heavy chain is the most variable and the primary determinant of antigen specificity.
The constant region determines the antibody’s effector function. It defines the isotype: IgM, IgD, IgG, IgA or IgE. Each isotype has different properties.
| Isotype | Function |
|---|---|
| IgM | First antibody produced. Pentameric. Strong complement activation. |
| IgD | Co-expressed with IgM on naive B cells. Role not fully understood. |
| IgG | Most abundant in blood. Long-lived. Crosses the placenta. |
| IgA | Dominant at mucosal surfaces. Secreted as a dimer. |
| IgE | Involved in allergy and parasite defence. Very low serum levels. |
V(D)J recombination
Section titled “V(D)J recombination”The enormous diversity of the antibody repertoire comes from V(D)J recombination. This process assembles the variable region gene from smaller gene segments during B cell development in the bone marrow.
The heavy chain variable region is assembled from three types of gene segments:
- V (variable): ~50 functional genes in humans
- D (diversity): ~25 functional genes
- J (joining): 6 functional genes
One V, one D and one J segment are randomly selected and joined together. The light chain uses only V and J segments.
Junctional diversity adds even more variation. At each junction, the recombination machinery deletes a few nucleotides and inserts random nucleotides called N-nucleotides and P-nucleotides. This happens at the V-D and D-J junctions.
The result is that the CDR3 region, which spans the V-D-J junction, is essentially random. The theoretical diversity of CDR3 sequences exceeds 10^13. This is far more than the number of B cells in a person.
Somatic hypermutation
Section titled “Somatic hypermutation”V(D)J recombination generates the initial antibody diversity. Somatic hypermutation (SHM) refines it.
After a B cell encounters its antigen in a germinal centre, the enzyme AID introduces point mutations throughout the variable region. The mutation rate is roughly one million times higher than the normal genome mutation rate.
Most mutations are neutral or harmful. But some improve antigen binding. B cells with improved binding receive survival signals and proliferate. B cells with worse binding die. This process is called affinity maturation.
Over multiple rounds of mutation and selection, antibodies become increasingly specific and high-affinity. You can measure affinity maturation by comparing the sequenced antibody to its predicted germline sequence. More mutations in the variable region means more maturation.
Replacement mutations change the amino acid. Silent mutations do not. Antigen-driven selection produces a higher ratio of replacement to silent mutations in the CDRs compared to the framework regions.
Isotype switching
Section titled “Isotype switching”Naive B cells express IgM and IgD. Upon activation, they can switch to IgG, IgA or IgE. This is called class switch recombination.
Isotype switching changes the constant region but leaves the variable region intact. The antibody keeps its antigen specificity but gains new effector functions.
BCR sequencing reveals the isotype distribution of the repertoire. This provides functional information:
- IgM dominance: early or primary immune response
- IgG dominance: mature, systemic immune response
- IgA enrichment: mucosal immune response
- IgE presence: allergic or anti-parasitic response
Clonotypes and clonal families
Section titled “Clonotypes and clonal families”B cells descended from the same V(D)J recombination event form a clone. All members of a clone share the same V gene, D gene, J gene and similar CDR3 sequence.
After activation, SHM creates mutations that distinguish individual members of the clone. These related but distinct sequences form a clonal family. You can reconstruct the evolutionary history of a clonal family as a phylogenetic tree, tracing how the antibody evolved from the germline sequence.
Clonal expansion occurs when a B cell proliferates in response to antigen. A single clone can grow to represent a large fraction of the total repertoire. Measuring clonal expansion reveals which antibodies are actively being produced.
Key clonal metrics include:
- Clonality: the fraction of the repertoire occupied by the top clones. High clonality means a few clones dominate.
- Diversity: Shannon or Simpson indices applied to clone sizes. Low diversity can indicate an antigen-driven response or a lymphoproliferative disorder.
- Convergent evolution: unrelated individuals producing similar CDR3 sequences against the same antigen. This suggests shared selective pressures.
Applications
Section titled “Applications”BCR sequencing is used across immunology and clinical research.
Vaccine development and evaluation. Track how the antibody repertoire responds to vaccination over time. Identify which V genes are recruited. Measure affinity maturation after booster doses.
Infectious disease. Characterise the antibody response to pathogens like SARS-CoV-2, HIV or influenza. Identify broadly neutralising antibodies. Track clonal evolution during chronic infection.
Autoimmune disease. Profile autoreactive B cells in diseases like lupus, rheumatoid arthritis and multiple sclerosis. Identify expanded clones targeting self-antigens.
Therapeutic antibody discovery. Mine the repertoire for antibodies with desired binding properties. BCR sequencing from immunised animals or convalescent patients is a source of candidate therapeutics.
Cancer immunology. Monitor minimal residual disease in B cell lymphomas and leukaemias by tracking the malignant clone. Characterise tumour-infiltrating B cells.
Transplant rejection. Identify donor-specific antibodies that mediate graft rejection.
Summary
Section titled “Summary”- BCR sequencing catalogues the antibodies present in a sample at single-sequence resolution.
- V(D)J recombination generates initial diversity. Somatic hypermutation refines it through affinity maturation.
- The CDR3 region of the heavy chain is the most variable part and determines antigen specificity.
- Isotype switching changes antibody function without changing specificity.
- Clonal analysis reveals which antibodies are actively expanding in response to immune challenges.
- Applications span vaccines, infectious disease, autoimmunity, cancer and therapeutic antibody discovery.