A novel method for high throughput TCR single cell VDJ-pairing with phenotypic analysis

Abstract

Abstract Single cell transcriptional analysis reveals a wealth of information that is obscured when analyzing the RNA of a sample en masse. Furthermore, it is important to know the identity of the TCR chain VDJ-rearrangement associated with the phenotypic state. This information can provide direct calculations of clonal frequency in various cell subsets, tracking of specific lymphocytes with treatment, and reveal paired information for both chains of the receptor for downstream Car-T development. Here, we discuss a novel method that identifies paired VDJ-rearrangements for both TCR alpha and beta chains from single cells barcoded via the BD Rhapsody Express single cell system. The phenotype of the cell is obtained using the BD Rhapsody RNA-seq kit, while the receptor information is amplified from the same cDNA using an iRepertoire-derived method incorporating a multiplex mix of primers associated with both the TCR alpha and beta loci. This targeted-seq strategy requires less read depth when compared to template-switch approaches. In one experiment, 3,000 TCR-beta unique CDR3 (uCDR3) were identified from ~7,000 barcoded cells using 1.5 million NGS reads with a 0.83 Pearson correlation upon repeat. Approximately 1,000 TCR alpha-beta paired uCDR3 were also called from the data. Additionally, the BD phenotyping results independently verify the immune repertoire data since the TCR beta chain constant gene overlays with the TCR-beta uCDR3 clonotypes for CD4+ and CD8+ subsets but is largely absent in the CD14+ monocyte population for both analyses. Thus, the developed technology provides the ability to simultaneously pair both alpha and beta chains for thousands of T-cells while obtaining information related to the functional status of each cell.