Immune Repertoire Sequencing

What is Immune Repertoire?

Adaptive immune system plays an extremely important role in counteracting against infectious pathogens and shielding the human body. The foundation of the adaptive immune system is fundamentally reliant upon the enormous diversity of T and B cell receptors (TCRs and BCRs) that can recognize epitopes from an astronomical number of different exogenous antigens and endogenous host factors.

Immune repertoire is known as the collection of functionally diverse B and T-cells in the circulatory system, which is a key measure of immunological complexity.T cells and B cells respectively orchestrate cellular and humoral immune responses in the organism. They each function via their surface T Cell Receptors (TCRs) and B Cell Receptors (BCRs) to recognize and bind to antigens, playing a critical role in eliminating pathogens or intrabody tumor cells. Operating independently, T cells and B cells mediate the body's complementary cellular and humoral immune responses, each utilizing their specific TCRs and BCRs on their surface. These receptors facilitate the identification and binding of antigens, thereby playing a fundamental role in the clearance of pathogens or endogenous tumor cells. The study of Immune repertoire is crucial to understand the response of adaptive immunity, and ultimately, sheds light on the discovery of novel infectious agents and holds the invaluable potential to aid in antibody or vaccine development, clinical diagnosis, treatment, and prevention.

High Throughput Immune Repertoire Sequencing

The tradition strategies for studying the immune repertoire, which includes spectratyping, Sanger sequencing et al., are laborious, costly, and are insufficient for generating a high-resolution picture of the immune repertoire. NGS (next generation sequencing) offers a powerful approach capable of analyzing the immune repertoire in a high-throughput manner. By providing enormous sequencing data, NGS has created an unprecedentedly high-resolution picture of the immune repertoire, which provides great potential for revealing dynamic changes in clonal populations during antigen stimulation.

Immune Repertoire Sequencing - Target Regions

In the realm of immune repertoire sequencing, the focus lies on T/B lymphocytes. Employing techniques such as multiplex PCR or 5’RACE, the objective is to amplify the complementary determining regions (CDR3) that determine the diversity of B cell receptors or T cell receptors. CDR3, being the most variable region, stands as the epicenter of exploration in immune repertoire sequencing studies. Its prominence is underscored by its status as the most extensively researched domain within the field.

Immune Repertoire SequencingIgG structure (Brian Douglas Weitzner 2015)

What is BCR Sequencing?

BCR sequencing employs high-throughput sequencing to detect the targeted amplification of BCR heavy and light chains. It comprehensively analyzes the base sequences resulting from BCR gene rearrangements, along with the abundance of each sequence. This technique finds application in investigating the transcriptional profiles and interrelationships of different B cell clones, delving into the functional specificity of B cells. Consequently, it aids in elucidating phenomena such as humoral immune response tolerance and the role of high-frequency mutations in abnormal antigen recognition by B cells.

The applications of BCR-Seq encompass:

Screening for somatic mutations in cellular populations of patients with primary immunodeficiency diseases like isolated IgA deficiency.

Guiding the treatment of autoimmune diseases (e.g., SLE, multiple sclerosis, type 1 diabetes), and monitoring disease progression during treatment.

Assessing the efficacy and tolerance of drug treatments for infectious diseases and malignant tumors.

Predicting and intervening in transplant rejection reactions, providing guidance on tolerance and rejection responses.

What is TCR Sequencing?

T-cell receptor sequencing (TCR-Seq) utilizes high-throughput sequencing (HTS) to detect the targeted amplification of T-cell antigen recognition determinant surface molecules. It analyzes their diversity, along with the TCR gene rearrangement base sequences and the abundance of each sequence before and after T-cell antigen recognition. This technique reflects changes in the cellular immune response mediated by T cells in physiological and pathological states. It is instrumental in studying the transcriptional profiles and interrelationships of different T cell clones, thereby unveiling deeper levels of T cell functional specificity.

Applications of TCR-Seq include:

Detection and guidance of post-tumor immunotherapy monitoring and treatment.

Evaluation of the effectiveness and tolerance of anti-infective immunotherapy.

Prediction and guidance of acute rejection in transplant rejection reactions.

Clinical trials and scientific research on autoimmune diseases.

Biomarkers and diagnostics for various infectious and neuroplastic diseases.

TCR/BCR Sequencing Service

CD Genomics has developed advanced TCR or BCR Sequencing strategy to amplify and sequence the immune repertoire with Next Generation Sequencing (NGS). Now, we are providing a comprehensive solution to evaluate the diversity of immune repertoire precisely in a cost-effective way by pooling the samples. We can tailor library construction and sequencing protocols to meet the specific requirements of different sample types and research projects, ensuring a personalized approach for our clients. Additionally, we integrate cutting-edge bioinformatics algorithms to facilitate visual data analysis, aligning with world-leading standards in computational biology.

Our Immune Repertoire Sequencing strategy is to capture the complementary determining region (CDR) of B-cell receptor (BCR) or T-cell receptor (TCR) using multiple-PCR or 5’ RACE methods, followed by deep sequencing. The workflow for the strategy is illustrated in the Figure below.

Schematics of steps for high-throughput sequencing of the Ig sequence repertoireSchematics of steps for high-throughput sequencing of the Ig sequence repertoire (Georgiou et al., 2014).

Selection of Two PCR Techniques

PCR Technique Multiplex PCR 5'RACE
Principle Same as regular PCR Rapid amplification of cDNA's 5' end in low-abundance transcripts
Amplification Region CDR3 CDR1-3
Sample Type DNA, RNA RNA (C region known required)
Advantages Simple operation, complete data Minimizes PCR amplification bias to a great extent
Disadvantages PCR amplification bias Laborious operation, loss of partial sequences, lower repeatability compared to multiplex PCR

Note: Choose different sequencing platforms and modes based on different needs.

Immune Repertoire Sequencing Process

Schematics of steps for high-throughput sequencing of the Ig sequence repertoire

Sample Requirement for Immune Repertoire Sequencing

DNA/RNA Sample Recommendations

Sample Type Total Amount Concentration RIN 285/185 Baseline & 5S Purity
Sorted T cells RNA/PBMCS RNA 2500 ng 235 ng/μL ≥7.0 ≥1.0 Smooth baseline, Normal 5S peak No DNA, protein/salt contamination, clear and non-viscous samples
Whole blood RNA/Tissue RNA ≥1 μg 270 ng/μL ≥7.0 ≥1.0 - -

Tissue Sample Recommendations

Tissue Type DNA Extraction Suggested Amount RNA Extraction Suggested Amount
Sorted Cells 21×10^6 ≥2×10^6
Fresh Animal Tissue Dry Weight ≥200 mg ≥30 mg
Whole Blood ≥2 mL -

Successful construction of TCR libraries can be achieved using samples obtained by immunomagnetic bead sorting of CD3-positive T cells and Ficoll-Paque gradient centrifugation separation of peripheral blood lymphocytes.

Our Technical Advantages

Comprehensive One-stop Service: We provide professional services covering the entire process, from experimental design, sample nucleic acid extraction and amplification, library construction, sequencing, information analysis, to data mining.

Cutting-edge Amplification Techniques: Utilizing multiplex PCR or 5'RACE technology, we employ primers optimized through plasmid adjustments, significantly reducing biases and achieving equivalent amplification for various clones.

Clonal Composition DistributionClonal Composition Distribution

V Gene Cluster HeatmapV Gene Cluster Heatmap

J Gene Cluster HeatmapJ Gene Cluster Heatmap

For Research Use Only. Not for use in diagnostic procedures.
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