Single-Cell VDJ Sequencing

Single-Cell VDJ Sequencing

1. Background

The immune repertoire refers to the entirety of T cell receptors (TCRs) and B cell receptors (BCRs) present within an individual at a given time point, reflecting the organism’s immune status and immune response capacity. TCRs and BCRs generate highly diverse antigen recognition sequences through V(D)J gene recombination, constituting the molecular basis by which the adaptive immune system recognizes and eliminates pathogens and tumor cells. In-depth analysis of the composition, diversity, and clonal dynamics of the immune repertoire is of significant importance for understanding immune response mechanisms and evaluating the efficacy of immunotherapy[1,2].

In the field of cell therapy, single-cell immune repertoire sequencing technology possesses unique value. For CAR-T cell products, it enables monitoring of endogenous TCR clonal composition and diversity changes; for TCR-T cell products, it enables verification of exogenous TCR expression and pairing accuracy; for TIL (tumor-infiltrating lymphocyte) therapy, it enables tracking of tumor-reactive T cell clone expansion and persistence[3,4].The advantage of single-cell VDJ sequencing lies in its ability to obtain complete TCR α/β chain or BCR heavy/light chain pairing information, overcoming the limitation of conventional bulk sequencing in determining chain pairing.

Generulor has established a comprehensive single-cell immune repertoire (TCR/BCR) sequencing and analysis platform, supporting end-to-end services from sample processing, single-cell VDJ library construction and sequencing, to specialized bioinformatics analysis, providing high-quality immune repertoire data and in-depth analytical support for immunology research and cell therapy product development.

2. Principles of Single-Cell Immune Repertoire Sequencing

The core principle of single-cell immune repertoire sequencing (single-cell VDJ sequencing) involves capturing and sequencing full-length variable region sequences of TCR or BCR at the single-cell level, while simultaneously obtaining transcriptomic information from the same cell. By tagging each cell with a unique molecular barcode, the pairing relationships of α/β chains (TCR) or heavy/light chains (BCR) originating from the same cell can be determined, enabling reconstruction of complete antigen receptor sequences.

The technical workflow comprises the following steps: first, individual immune cells are encapsulated within gel bead-containing droplets with cell barcodes using single-cell platforms such as 10x Genomics, followed by cell lysis and mRNA capture; subsequently, universal sequences are appended to the cDNA termini through reverse transcription and template-switching reactions; enrichment amplification is performed using primers specific to the TCR/BCR constant regions, while preserving 5’ end transcriptomic information; after library construction and high-throughput sequencing, specialized bioinformatics analysis pipelines are employed for VDJ sequence assembly, clonotype identification, pairing analysis, and diversity assessment.

Figure 1. Schematic workflow of single-cell immune repertoire (TCR/BCR) sequencing technology

3. Technological Innovations and Advantages of Single-Cell Immune Repertoire Sequencing

3.1 Core Technological Innovations

3.1.1 Complete Chain Pairing Information Acquisition

Overcoming the limitations of conventional bulk sequencing, precise receptor chain pairing analysis is achieved:

(1) Determination of the native pairing relationships of TCR α/β chains or BCR heavy/light chains at the single-cell level;

(2) Acquisition of complete V(D)J rearrangement sequences, including CDR3 and framework regions;

(3) Support for exogenous TCR expression verification and pairing accuracy analysis in TCR-T products.

3.1.2 Multi-Modal Integrated Analysis

Integration of immune repertoire with transcriptomic/proteomic information for in-depth immune characterization:

(1) Simultaneous acquisition of VDJ sequences and gene expression profiles from the same cell;

(2) Support for combined analysis with CITE-seq (protein expression);

(3) Correlation of clonotypes with cell phenotype, functional status, and differentiation trajectories.

3.1.3 Clonal Tracking and Dynamic Monitoring

Supporting longitudinal studies to track dynamic changes in immune clones:

(1) Identification and tracking of expansion and contraction of antigen-specific reactive clones;

(2) Assessment of immune repertoire diversity and compositional changes before and after treatment;

(3) Monitoring of clonal dynamics and persistence following CAR-T/TCR-T product infusion.

3.1.4 Antigen Specificity Prediction

Prediction of TCR/BCR antigen specificity based on sequence features:

(1) Alignment and annotation against databases of known antigen-specific TCR/BCR sequences;

(2) Prediction of potential antigen targets using machine learning approaches;

(3) Identification of tumor neoantigen-reactive T cell clones.

3.2 Methodological Validation and Performance Metrics

Generulor has conducted comprehensive systematic validation of the single-cell immune repertoire sequencing platform to ensure data quality and analytical accuracy:

4. Application Scenarios and Service Advantages

4.1 Application Scenarios

Single-cell immune repertoire sequencing technology has broad applications across multiple fields:

(1) Quality assessment of CAR-T/TCR-T cell products: analysis of endogenous TCR clonal composition and verification of exogenous TCR expression and pairing;

(2) TIL therapy research: identification and tracking of tumor-reactive T cell clones, assessment of TIL diversity and functional status;

(3) Immune checkpoint inhibitor efficacy evaluation: monitoring T cell clonal expansion and immune repertoire dynamic changes before and after treatment;

(4) Vaccine and antibody development: analysis of vaccine-induced B cell responses and screening of high-affinity antibody sequences;

(5) Autoimmune disease research: identification of autoreactive T/B cell clones and elucidation of disease pathogenesis;

(6) Infectious immunity research: tracking pathogen-specific immune responses and memory cell formation.

4.2 Service Advantages

(1) Comprehensive solution: providing one-stop services from sample processing, VDJ library construction and sequencing, to specialized analysis;

(2) Multi-modal integration: supporting combined VDJ, transcriptomic, and proteomic analysis for comprehensive immune characterization;

(3) Accredited quality management: the laboratory operates under both ISO 9001 quality management system and ISO/IEC 17025 accreditation standards;

(4) Specialized analytical team: extensive experience in immune repertoire data analysis and cell therapy research;

(5) Customized services: supporting personalized analytical needs including antigen specificity analysis and clonal tracking.

5. Exemplary Report of Single-Cell Immune Repertoire Sequencing

Generulor provides comprehensive single-cell immune repertoire sequencing analysis reports, encompassing foundational information including sequencing data quality assessment and VDJ detection statistics.

Figure 2 illustrates the bioinformatics analysis pipeline:VDJ-seq data analysis encompasses three core steps: first, quality control and preprocessing of raw FASTQ data, including removal of low-quality and germline sequences; then, sequence deduplication using unique molecular identifiers (UMIs) to generate consensus sequences and eliminate PCR amplification bias; finally, V(D)J gene segment annotation and CDR3 identification using IgBlast, followed by immunoglobulin clonotype assembly and output of standardized result files, ensuring analytical accuracy and reproducibility.

Figure 2. Single-Cell Transcriptome (VDJ) Analysis Pipeline Schematic

The core contents of the report include:

(1) VDJ Sequence Assembly and Annotation Analysis:Statistical summary of quality metrics for each sample, including VDJ detection rate, proportion of productive sequences, and chain pairing rate, along with V/D/J gene usage frequency analysis and CDR3 length distribution.

Figure 3. VDJ Gene Usage Frequency and CDR3 Length Distribution

(2) Clonotype Identification and Frequency Analysis:Clonotype definition based on CDR3 sequences, statistical summary of cell counts and frequencies for each clonotype, identification of dominant and rare clonotypes, and generation of clonotype frequency distribution plots.

Figure 4. Clonotype Frequency Distribution and Top Clonotype Display

(3) Immune Repertoire Diversity Analysis:Calculation of standard diversity metrics including Shannon diversity index, Simpson index, and clonality, assessing the richness and evenness of the immune repertoire.

Figure 5. Immune Repertoire Diversity Index Statistics

(4) Chain Pairing and Receptor Reconstruction Analysis:Visualization of TCR α/β chain or BCR heavy/light chain pairing relationships, reconstruction of complete antigen receptor sequences, supporting subsequent functional validation experiments.

Figure 6. TCR/BCR Chain Pairing Sankey Diagram Analysis

(5) Clonal Sharing and Differential Analysis:Comparison of clonotype sharing and differences across samples or time points, tracking dynamic changes of specific clones, and assessment of immune response specificity.

Figure 7. Inter-Sample Clonal Sharing Venn Diagram and Tracking Analysis

(6) VDJ-Transcriptome Integrated Analysis:Integration of VDJ sequences with transcriptomic data, correlating clonotypes with cell phenotype and functional status, and identification of clonal subpopulations with specific phenotypic features.

Figure 8. VDJ-Transcriptome Integrated UMAP Clustering Analysis

6. Service Scope for Single-Cell Immune Repertoire Sequencing

*Turnaround time: standard workflow30-40 business days;

7. Sample Requirements

*Note: (1) All samples must meet the quality standards described above; (2) Cell sorting enrichment is recommended when T/B cell proportions are low; (3) For special sample types, please consult with the Generulor technical team in advance (Tel: 400-6309596; Product ordering/Technical support: service@generulor.com).

8. References

[1] Peter Chovanec. (2018). Unbiased quantification of immunoglobulin diversity at the DNA level with VDJ-seq. Nat Med, VOL.13 NO.6.

[2] Georgiou G, et al. (2014). The promise and challenge of high-throughput sequencing of the antibody repertoire. Nat Biotechnol, 32(2):158-168.

[3] Yost KE, et al. (2019). Clonal replacement of tumor-specific T cells following PD-1 blockade. Nat Med, 25(8):1251-1259.

[4] Zheng C, et al. (2021). Landscape of Infiltrating T Cells in Liver Cancer Revealed by Single-Cell Sequencing. Cell, 169(7):1342-1356.