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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 CSV files² 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. We recommend uploading the filtered feature barcode matrices as either the Hierarchical Data Format (H5 or HDF5)⁴ or Market Exchange Format (MEX)⁵. 

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).

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Analyzing the Single cell V(D)J node

Summarize V(D)J

• Under QA/QC tasks is the Summarize V(D)J task which will summarize the V(D)J contents by Sample name, # Cells, Barcode count, Clonotypes, Variable genes, Diversity genes, Joining genes, and Constant genes. 

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Clonotype Frequency Plot

Under Exploratory Analysis tasks is the Clonotype Frequency Plot task which will summarize the V(D)J node into plots of interest in the Data Viewer. Similar manual comparisons can be made in the Data Viewer. These may include determining the T cell receptor and B cell receptor chains that make up clonotypes in the samples, quantifying the clone diversity by frequency, comparing the immune repertoire between samples, and visualizing clones and gene expression data together on scatterplots like a UMAP. 

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