Variable (V), Diversity (D), and Joining (J) recombination analysis

V(D)J recombination occurs in lymphocytes when T and B cells assemble variable (V), diversity (D), and joining (J) gene segments, contributing to the generation of receptors which recognize and respond to perturbations. V(D)J recombination produces clones of unique T cell receptor (TCR) chains or B cell receptor (BCR) chains giving rise to the diverse repertoire of T and B cell populations which are imperative to adaptive immune system function¹. The frequency of generated clones can be measured and explored, giving researchers a powerful view into variation, expansion, and diversity within the biological system. You can import filtered contig annotation files in csv file format² from the 10X Genomics Cell Ranger V(D)J or multi pipeline³. If there is matching gene expression data, it can also be imported and analyzed within the same project.

Terminology 

• UMI (Unique Molecular Identifier): random 10 bp nucleotide sequence that distinguishes which reads came from the same transcript. 
• Barcode: the unique identifier in each droplet which usually contains reads from a single cell. 
• Contig: assembled reads containing the same UMI and barcode that align to the same transcript sequence. 
• Complementarity-Determining Region: CDR1, CDR2, and CDR3 are important in antigen binding of a T or B cell receptor. 
• CDR3 (Complementarity-Determining Region 3): CDR3 spans the V(D)J junction. There is one CDR3 nucleotide sequence for each V(D)J contig.
• Clonotype: cells derived from a common ancestor during clonopoiesis which have a particular composition. The cells in a clonotype can have a different number of chains or different CDR3 regions but still be considered a single clone (CDR3 is a highly variable region used for binding; an example of different CDR3 regions would be from affinity maturation which can occur in memory B cells).

Understanding Clone Composition

Multiple cells can have the same clonotype and each clonotype can have multiple makeups. Each clonotype contains one or more chains (TRA and TRB for T cells and IGH, IGK and IGL for B cells), the highest scoring V, D, and J gene segments, and CDR3 nucleotide sequence. T cells have a TRA and TRB chain with V, D, J and C regions.  In B cells, IGH is the heavy chain which has a V, D, and J region while IGK and IGL are the light chains with a V and J region. The Immunoglobulins have two identical heavy chains and two identical light chains. B cell isotypes are antigenic determinants that characterize the classes and subclasses of heavy chains and types and subtypes of light chains; the constant region (C gene) produced by the B cell changes but the V regions and specificity do not.  Constant regions do not participate in antigen recognition, instead C regions interact to mediate biological function; so isotypes have different function but can bind the same antigen. 

References

1. Tonegawa, S. Somatic generation of antibody diversity. Nature 302,575–581 (1983). https://doi.org/10.1038/302575a0
2. https://support.10xgenomics.com/single-cell-vdj/software/pipelines/latest/output/annotation#contig-annotation 
3. https://support.10xgenomics.com/single-cell-vdj/software/overview/welcome