Once the libraries are sequenced, it is possible to pair the alpha and beta sequences from each individual cell. The 10x Genomics Cell Ranger bioinformatics pipeline assembles V(D)J short sequence reads into consensus alpha and beta chain annotated full-length paired V(D)J profiles.

The Cell Ranger pipeline filters the reads based on shared homology with germline V, D, J, constant segments and assembles the filtered reads within each barcode producing contigs, then annotates the contig sequences with the best germline V, D, J, constant, and UTR matches, detecting and translating the CDR3 sequence. It then groups cells into clonotypes, which share all productive CDR3 sequences, building a consensus for each chain in each clonotype.

To validate the Chromium Single Cell V(D)J Solution performance, the product was used to profile a variety of samples containing T cells. In one experiment, two samples of peripheral blood mononuclear cells (PBMCs) from the same healthy individual were sequenced to confirm that the two independently run samples would exhibit similar behavior. Since the samples came from a healthy individual with no known challenges to the immune system, researchers expected to see high T-cell diversity and low antigen specificity.

Cell Ranger software grouped the T cells into clonotypes and calculated the percent that each clonotype was represented in the sample. In the first sample, 2,809 clonotypes were detected, and 2,949 were detected in the second sample. As expected, no clonotype made up >0.5% of either sample, demonstrating a very high diversity and low specificity in the sample.

To determine antigen specificity, an experiment was performed using T cells exposed to the Epstein-Barr Virus (EBV) in cell culture (Figure 3). The EBV-specific T cells captured and sequenced by the Chromium System were sorted into clonotypes. It was found that 55% of the sequenced T cells shared one major alpha and beta chain, TRAV12-3:J20 (CDR3: CATQGSNDYKLSF), TRBV9:D1:J1-4 (CDR3: CASSTGQVATNEKLFF); 9% shared a second, unrelated clonotype, TRAV5:J15, TRBV14:D2:J2-1; 4% had two related clonotypes that shared a common beta chain3% with TRAV5:J15, TRBV29-1:D1:J1-4 and 1% with TRAV5:J23, TRBV29-1:D1:J1-4.

After the antigen specificities and frequencies of each of the four most dominant clonotypes were determined, limit-of-detection (LOD) experiments were performed using 1:99 dilutions of the EBV-specific T cells mixed into replicate samples of PBMCs from a healthy donor (Figure 3).

In this experiment, one would expect the most dominant clonotype (55%) from the EBV-specific T cells to be observed at a frequency of 0.55% when spiked into the PBMC background. Consistent with these expectations, 16 cells (0.4%) and 7 cells (0.3%) were found to express the major EBV-specific clonotype (TRAV12-3:J20, TRBV9:D1:J1-4) in the first and second spike-in replicates, respectively.

Interestingly, the Chromium V(D)J Solution was able to detect two cells (0.05%) and one cell (0.05%), respectively, of the second most abundant EBV-specific clonotype, resulting in an LOD of <0.1%. This limit of detection is likely to be pushed even lower as future experiments using more input T cells and greater sequencing depths enable the detection of even more rare known clonotypes.

The V(D)J Solution supports diverse basic and translational research studies of applied immunology and will ultimately accelerate our understanding of human health and disease. Particularly exciting application areas that will be propelled by the V(D)J Solution include T cellbased immunotherapies and with the addition of a planned B-cell-specific VDJ solution, vaccine development.

The V(D)J Solution will do this by enabling the identification of the true paired diversity of antigen receptors on a single-cell basis and thereby more effectively enable functional studies into the molecular genetic determinants of antigen specificity.

When coupled with assessments of immune repertoire diversity across experimental contexts of normal healthy tissues, longitudinal or case/control studies, and shared immune responses to common exposure histories, the V(D)J Solution will elucidate the adaptive immune system with greater resolution than ever before

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