Table of Contents

What Is TCR and BCR Immune Repertoire Sequencing?

T-cell receptors (TCRs) and B-cell receptors (BCRs) are the molecular sensors of adaptive immunity. Each T or B cell carries a unique receptor generated through V(D)J recombination — a process that rearranges variable (V), diversity (D), and joining (J) gene segments to create a diverse repertoire capable of recognizing a vast array of antigens. TCR and BCR immune repertoire sequencing profiles this collection of receptors in a sample, revealing the composition, diversity, and clonal architecture of the adaptive immune response. The complementarity-determining region 3 (CDR3) — the most variable part of each receptor — serves as a molecular tag for individual T- and B-cell clones.

What this service provides:

5'RACE-based library preparation — Captures the complete V(D)J variable region, including CDR1, CDR2, and CDR3
Illumina next-generation sequencing — TCR transcripts (TRA/TRB) and BCR transcripts (IGH/IGK/IGL)
IMGT-aligned clonotype annotation — Standardized nomenclature against the international IMGT reference database
Quantitative repertoire metrics — Clonality, diversity (Shannon, Simpson), V-J gene usage, CDR3 length distribution

IMGT-based V(D)J annotation principle for TCR/BCR immune repertoire sequencing

IMGT-based V(D)J annotation: reads aligned to the international IMGT reference database for standardized clonotype identification.

The IMGT database (imgt.org) is the international reference for immunoglobulin and T-cell receptor gene annotation, providing standardized clonotype nomenclature across studies [1].

5'RACE vs Multiplex PCR — Method Comparison

Two main approaches are used for TCR/BCR library preparation: 5' Rapid Amplification of cDNA Ends (5'RACE) and multiplex PCR (mPCR). Choosing the right method affects which receptor regions are captured and how faithfully the repertoire is represented.

Dimension	5'RACE	Multiplex PCR
Region captured	Complete V(D)J (CDR1, CDR2, CDR3)	CDR3 only
Amplification bias	Low — single primer pair with universal adapter	Moderate to high — multiple primer pairs with varying efficiencies
Quantification accuracy	Higher — fewer amplification rounds, less primer competition	Lower — primer-specific biases can distort clone frequencies
RNA input requirement	~1–100 ng total RNA	Typically requires more starting material
Chain coverage	TRA, TRB, TRG, TRD; IGH, IGK, IGL	Variable by panel design
Best suited for	Comprehensive repertoire profiling, full-length clonotype analysis, low-input samples	Targeted CDR3 screening, fixed-panel studies

Why we use 5'RACE: The 5'RACE method adds a universal adapter to the 5' end of cDNA during reverse transcription, enabling amplification with a single primer pair instead of a multiplex primer pool. This reduces amplification bias and captures the full V(D)J region — CDR1, CDR2, and CDR3 — rather than just the CDR3 loop. Complete V(D)J information supports V(D)J gene usage analysis, somatic hypermutation detection in BCR, and more accurate clonotype identification [2].

Multiplex PCR is a viable alternative when only CDR3-level information is needed, but it introduces primer-specific amplification bias that can skew clone frequency estimates. For studies requiring accurate clonal abundance, full V(D)J annotation, or low-input capability, 5'RACE delivers more complete and quantitative results.

RNA vs DNA Input — Selection Guide

TCR/BCR repertoire sequencing can start from RNA or genomic DNA (gDNA). Each has distinct implications for what the data represents.

Dimension	RNA Input	gDNA Input
What is measured	Expressed TCR/BCR transcripts	Genomic V(D)J rearrangements
Clone frequency	Reflects expression level — one cell may produce multiple transcript copies	One template per cell — closer to actual clone size
CDR region coverage	Full V(D)J (CDR1, CDR2, CDR3) with 5'RACE	Typically CDR3 only — introns between V/D/J segments limit full-length PCR
Somatic hypermutation (SHM)	Detectable in BCR transcripts	Detectable but requires more complex analysis
Input requirement	~1–100 ng total RNA	Typically ≥2 µg gDNA
FFPE compatibility	Challenging — RNA degrades in FFPE	More suitable — DNA is relatively stable in FFPE

Guidance:

Choose RNA for comprehensive repertoire profiling with full V(D)J annotation, SHM analysis, and when working with limiting input material.
Choose gDNA for archival FFPE samples, when avoiding transcript-level expression bias is critical, or when only CDR3-level clonality assessment is needed.

Our standard service uses RNA input with 5'RACE. A gDNA-based option is available upon consultation.

TCR and BCR Sequencing Workflow

The service follows six connected steps, with quality control at key transitions.

TCR and BCR immune repertoire sequencing workflow — 6 steps from sample QC to data delivery

1. Sample Receipt and QC

Total RNA is extracted (when starting from cells or tissue) and assessed for concentration (≥10 ng/µL), purity (A260/A280 ≥1.8), and integrity (RIN ≥7 recommended). Samples passing QC proceed to library preparation.

2. Library Preparation (5'RACE)

Reverse transcription primes from the constant region of TCR (TRAC/TRBC) or BCR (IGH/IGK/IGL) transcripts. A universal adapter is ligated to the 5' end of cDNA, enabling single-primer-pair amplification of the complete V(D)J region. Transcript-specific barcodes may be incorporated for duplicate marking and error correction.

3. Sequencing

Libraries are sequenced on the Illumina platform (MiSeq PE300 or HiSeq PE150/250 configuration). Paired-end reads span the CDR3 region and extend into V and J gene segments for complete clonotype assembly.

4. Basecalling and Raw Data QC

Raw sequencing reads are filtered by quality score (Q30 target), trimmed of adapter sequences, and checked for read-pair concordance. Read counts per sample are recorded.

5. IMGT Alignment and Clonotype Assembly

Quality-filtered reads are aligned to the IMGT reference database for V, D, and J gene assignment. CDR3 nucleotide and amino acid sequences are identified, and clonotypes are assembled by unique CDR3 sequence + V-J combination.

6. Repertoire Analysis and Data Delivery

Clonotype tables are processed for diversity metrics, V-J gene usage, CDR3 length distribution, and clonality assessment. Results are compiled into a structured report and delivered with raw data files.

QC checkpoint at each transition: sample QC → library QC (concentration, size distribution) → sequencing QC (Q30, cluster density) → alignment QC (alignment rate, clonotype count) → report QC.

Sample Requirements

The following table summarizes standard sample types and submission guidelines. Contact our team for project-specific recommendations.

Sample Type	Recommended Input	Minimum Input	Purity / Quality	Shipping Condition
Total RNA (from T/B cells)	≥100 ng	10 ng	A260/A280 ≥1.8, RIN ≥7	Dry ice
Peripheral blood	5–10 mL	2 mL	EDTA or citrate tube	Cold pack (4°C), within 24 h
PBMC	≥2×10⁵ cells	1×10⁵ cells	Viability ≥80%	Dry ice (frozen) or cold pack (fresh, within 24 h)
Fresh frozen tissue	50–100 mg	20 mg	Snap-frozen within 30 min of collection	Dry ice
gDNA	≥2 µg	500 ng	A260/A280 ≥1.8, concentration ≥20 ng/µL	Dry ice or cold pack
FFPE tissue	5–10 scrolls (10 µm)	3 scrolls	Assessed at intake	Ambient

RNA samples: include RIN or gel image if available.
Blood samples: process within 24 hours of collection for optimal lymphocyte viability.
FFPE samples: RNA degradation is expected; gDNA-based TCR/BCR analysis may be more suitable. Contact us before submission.

Bioinformatics Analysis

Standard Analysis — Every TCR/BCR sequencing project includes:

Raw data QC — Read quality filtering, adapter trimming, read-pair merging
IMGT alignment — V, D, J gene assignment against the IMGT reference database [1]
CDR3 identification — Nucleotide and amino acid CDR3 sequence calling
Clonotype assembly — Clustering by unique CDR3 + V-J gene combination
Clonotype frequency profiling — Rank-abundance table of all detected clonotypes
V(D)J gene usage — V gene, J gene, and V-J pairing frequency analysis
CDR3 length distribution — Amino acid length histogram per chain
Diversity assessment — Shannon entropy, Simpson index, inverse Simpson index, clonality score
Report generation — Integrated analysis report with figures, tables, and methods documentation

Optional Add-on Analyses:

Longitudinal clonotype tracking — Track specific clonotypes across multiple timepoints; identify expanding or contracting clones
Sample group comparison — Differential V-J usage, diversity comparison, shared clonotype analysis between groups
Somatic hypermutation (SHM) analysis — Mutation frequency and pattern analysis for BCR sequences (IgG/IgM)
CDR3 physicochemical property analysis — Hydrophobicity, charge distribution, amino acid composition profiling

For custom analysis requirements or larger-scale projects, see our bioinformatics analysis service page.

Deliverables

Each TCR/BCR sequencing project includes raw data, analysis outputs, visual results, and documentation.

Category	Contents
Raw data	FASTQ files (demultiplexed, quality-filtered); Sequencing QC report (Q30 scores, read counts, cluster density)
Analysis outputs	Clonotype frequency tables (Excel and tab-delimited format); IMGT alignment summary (V/D/J gene assignment per clonotype); CDR3 nucleotide and amino acid sequences
Visual results	CDR3 length distribution plot; V-J gene usage heatmap; Diversity metric bar charts (Shannon, Simpson, clonality); Clonotype rank-abundance curve
Documentation	Full analysis report (PDF) with methods, parameters, and interpretation notes; Project metadata file (sample IDs, sequencing configuration, analysis software versions)

All files are delivered via secure data transfer or hard drive, per customer preference.

Applications of TCR and BCR Sequencing

Cancer Immunology

Profile tumor-infiltrating lymphocyte (TIL) repertoires to assess anti-tumor immune responses, identify expanding T-cell clones, and correlate repertoire features with immunotherapy outcomes. BCR analysis reveals intra-tumoral B-cell responses and antibody repertoire shifts.

Infectious Disease Research

Track antigen-specific T- and B-cell clonal expansion during acute infection and convalescence. Identify public clonotypes shared across individuals responding to the same pathogen.

Vaccine Development

Assess vaccine-induced clonal expansion, repertoire diversification, and memory B-cell persistence. Compare pre- and post-vaccination repertoires to evaluate immunogenicity and durability.

Autoimmune Disease

Characterize T- and B-cell repertoire skewing in autoimmune conditions. Identify disease-associated clonotypes and assess repertoire changes following immunomodulatory therapy.

Transplantation

Monitor donor-reactive T-cell clones and assess repertoire reconstitution following hematopoietic stem cell transplantation or solid organ transplant.

Related: HLA typing service

Biomarker Discovery

Identify repertoire-level signatures — clonality, diversity, specific V-J usage patterns — that correlate with disease state, treatment response, or prognosis across patient cohorts.

Reference

Giudicelli V, Chaume D, Lefranc MP. IMGT/GENE-DB: a comprehensive database for human and mouse immunoglobulin and T cell receptor genes. Nucleic Acids Research. 2005;33(Database issue):D256–D261. https://doi.org/10.1093/nar/gki010
Rosati E, Dowds CM, Liaskou E, Henriksen EKK, Karlsen TH, Franke A. Overview of methodologies for T-cell receptor repertoire analysis. BMC Biotechnology. 2017;17(1):61. https://doi.org/10.1186/s12896-017-0379-9
Frank ML, Lu K, Erdogan C, et al. T-cell receptor repertoire sequencing in the era of cancer immunotherapy. Clinical Cancer Research. 2023;29(6):994–1008. https://doi.org/10.1158/1078-0432.CCR-22-2469
Li R, Wang J, Li X, et al. T-cell receptor sequencing reveals hepatocellular carcinoma immune characteristics according to Barcelona Clinic liver cancer stages within liver tissue and peripheral blood. Cancer Science. 2024;115(1):94–108. https://doi.org/10.1111/cas.16013
Xie S, Yan R, Zheng A, Shi M, et al. T cell receptor and B cell receptor exhibit unique signatures in tumor and adjacent non-tumor tissues of hepatocellular carcinoma. Frontiers in Immunology. 2023;14:1161417. https://doi.org/10.3389/fimmu.2023.1161417
Bolotin DA, Poslavsky S, Mitrophanov I, et al. MiXCR: software for comprehensive adaptive immunity profiling. Nature Methods. 2015;12(5):380–381. https://doi.org/10.1038/nmeth.3364

Demo Results

Below are representative data types generated during a standard TCR/BCR immune repertoire sequencing project using the 5'RACE workflow. All figures are illustrative examples; results vary by sample type, species, and experimental conditions.

CDR3 amino acid length distribution for TRB chain

Figure 1: CDR3 Length Distribution
A Gaussian-like distribution of CDR3 amino acid lengths, typically centered around 12–16 amino acids for TRA/TRB chains. This distribution reflects the natural V(D)J recombination process and serves as a quality indicator. Representative figure type as described in Frank et al. 2023.

Figure 2: V-J Gene Usage Heatmap
Two-dimensional heatmap with V gene segments (rows) and J gene segments (columns). Color intensity represents the frequency of each V-J pairing. Uneven V-J usage is expected and reflects thymic selection, MHC restriction, and antigen-driven expansion.

Repertoire diversity metrics — Shannon entropy, Simpson index, clonality score

Figure 3: Clonality and Diversity Metrics
Bar chart comparing Shannon entropy, Simpson index, and clonality score across sample groups. High Shannon entropy indicates a polyclonal repertoire; high clonality indicates oligoclonal expansion — common in tumor-infiltrating lymphocytes or post-vaccination responses.

Reference

Frank ML, Lu K, Erdogan C, et al. T-cell receptor repertoire sequencing in the era of cancer immunotherapy. Clinical Cancer Research. 2023;29(6):994–1008. https://doi.org/10.1158/1078-0432.CCR-22-2469

TCR and BCR Sequencing FAQ

1. Which chains do you cover?

For TCR: TRA (α), TRB (β), TRG (γ), and TRD (δ). For BCR: IGH (heavy chain), IGK (kappa light chain), and IGL (lambda light chain). Multiple chains can be amplified in the same 5'RACE reaction.

2. Can you sequence both TCR and BCR from the same sample?

Yes. The 5'RACE workflow can amplify TCR and BCR transcripts from a single RNA sample. This is efficient when both T-cell and B-cell repertoire information is needed, such as in tumor microenvironment or vaccine response studies.

3. What sequencing depth do I need?

For standard clonality and diversity assessment, 2–5 million reads per chain is typically sufficient. For rare clonotype detection or in-depth repertoire characterization, higher depth may be recommended. Our team can advise based on your study design.

4. What species do you accept?

Human and mouse are standard. Other species (rat, non-human primate, etc.) can be accommodated with customized primer design. Contact us to discuss your species of interest.

5. Can I submit FFPE samples?

FFPE samples can be submitted for gDNA-based TCR/BCR analysis. RNA-based 5'RACE may have reduced success with FFPE due to RNA degradation. We recommend contacting us before FFPE submission so we can advise on the most suitable approach.

6. How do I choose between RNA and gDNA input?

RNA input (5'RACE) captures complete V(D)J regions (CDR1/2/3) and reflects transcript-level expression — suitable for comprehensive profiling. gDNA input avoids expression-level bias (one template per cell) but is typically limited to CDR3-level analysis. Choose based on your research question: transcript-level repertoire with full annotation (RNA) or cell-level quantification with CDR3 focus (gDNA).

7. Can clonotypes be tracked across multiple timepoints?

Yes. Clonotype tracking across longitudinal samples is available as an optional add-on. Clonotypes from different timepoints are matched by CDR3 nucleotide sequence identity, and frequency changes are quantified to identify expanding or contracting clones.

8. What bioinformatics tools do you use for repertoire analysis?

Our standard pipeline uses MiXCR for clonotype assembly and IMGT/HighV-QUEST for V(D)J gene annotation against the IMGT reference database. Custom analysis scripts handle additional metrics, visualization, and longitudinal tracking.

Case Study: Immune Repertoire Signatures in Hepatocellular Carcinoma

Open Access Publication Highlight

T cell receptor and B cell receptor exhibit unique signatures in tumor and adjacent non-tumor tissues of hepatocellular carcinoma

Journal: Frontiers in Immunology (IF 5.7)
Published: 2023
License: CC BY 4.0

Background

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide. The landscape of infiltrating T and B cells and their receptor repertoires in HCC remains incompletely characterized. Understanding how TCR and BCR features differ between tumor and adjacent non-tumor tissue can inform immunotherapy strategies and biomarker development.

Methods

Xie, Yan, Zheng, Shi et al. (2023) performed multi-omics profiling of paired tumor and adjacent non-tumor liver tissues from 64 treatment-naive HCC patients. The study combined bulk TCR/BCR sequencing (5'RACE + Illumina HiSeq3000 PE150, processed with MiXCR v3.0.13), bulk RNA-seq, whole-exome sequencing, and HLA sequencing. Repertoire richness was assessed by unique clonotype count; evenness by normalized Shannon diversity entropy (NSDE); similarity between paired samples by the Morisita-Horn similarity index (MHSI).

Results

TCR and BCR features differed between tissue compartments. BCR IgH and TRG richness and evenness were significantly higher in non-tumor tissues versus paired tumor tissues. BCR somatic hypermutation (SHM) was also higher in non-tumor tissues, indicating sustained B-cell responses outside the tumor.
BCR repertoires diverged more between compartments than TCR repertoires. T-cell clones were more evenly distributed across both compartments, while B-cell clones showed stronger compartment-specific expansion.
Immune repertoire features declined with HCC progression. TRB richness and evenness decreased from early-stage (TNM 1) to advanced-stage (TNM 2+) tumors. Conversely, BCR SHM intensified in advanced-stage disease.
Repertoire metrics correlated with survival. Higher immune repertoire evenness in tumor tissues and lower TCR richness in non-tumor tissues were associated with better overall survival, progression-free survival, and recurrence-free survival.

Figure from Xie et al. 2023 — TCR and BCR immune repertoire features in HCC tumor and non-tumor tissues

TCR and BCR repertoire features distinguish tumor and adjacent non-tumor tissues in HCC. Adapted from Xie et al., Frontiers in Immunology, 2023, under CC BY 4.0 license.

Conclusion

This study demonstrated that bulk TCR/BCR immune repertoire sequencing resolves compartment-specific immune signatures in HCC — distinguishing tumor from non-tumor tissue, tracking repertoire changes with disease progression, and identifying features associated with patient prognosis. The results highlight the value of simultaneous TCR and BCR profiling for cancer immune microenvironment characterization and biomarker discovery.

Reference

Xie S, Yan R, Zheng A, Shi M, et al. T cell receptor and B cell receptor exhibit unique signatures in tumor and adjacent non-tumor tissues of hepatocellular carcinoma. Frontiers in Immunology. 2023;14:1161417. https://doi.org/10.3389/fimmu.2023.1161417