TCR Multiplex PCR Sequencing

TCR Multiplex PCR Sequencing

1. Background

T cell receptor (TCR) diversity is the molecular foundation of adaptive immune responses. Its clonal composition and functional status are closely linked to the onset, progression, and treatment outcomes of tumor immunity, autoimmune diseases, and infectious diseases. Among conventional bulk TCR sequencing approaches, multiplex PCR directly amplifies the rearranged CDR3 region using a mature primer design system, is compatible with multiple sample formats including genomic DNA (gDNA) and FFPE, and offers significant advantages in the robust quantification of clonal frequencies. It is the most widely accepted technical route from a regulatory standpoint for hematologic malignancy clonality assessment and minimal residual disease (MRD) monitoring studies.

In cutting-edge drug development and translational medicine, the value of multiplex PCR–based TCR sequencing has gained broad recognition. The FDA guidance Hematologic Malignancies: Regulatory Considerations for Use of Minimal Residual Disease (2020) lists NGS-based TCR/Ig rearrangement detection as one of the methods for MRD assessment; the NGS standardization framework developed by the EuroClonality-NGS consortium on the basis of the classic BIOMED-2 primer system has further established this route's authoritative international standing in clonality determination. For cell therapy products such as TCR-T/CAR-T, the FDA's Considerations for the Development of CAR T Cell Products (2024) and China's CDE Technical Guidelines for Clinical Trials of Immune Cell Therapy Products (Trial) (2021) likewise set out clear requirements for T cell clonality amplification and product characterization.

ZhuHai GeneRulor provides end-to-end services spanning sample QC, multiplex PCR library preparation, high-throughput sequencing, and systematic bioinformatic analysis, helping clients precisely resolve T cell clonal dynamics in a robust and cost-effective manner—delivering high-quality data support for immune monitoring, MRD studies, and the research characterization of cell therapy products.

2. Technical Principle

Figure 1. Workflow of Multiplex PCR Amplification of the TCR Rearrangement Region

The workflow comprises the following core steps:

（1）Nucleic Acid Extraction

Genomic DNA (gDNA) or total RNA is extracted from the sample of interest, followed by quality assessment and quantification. The DNA-based approach offers broader sample compatibility, supporting FFPE and partially degraded samples; the RNA-based approach requires a qualified RIN value and allows the introduction of UMIs to further correct for amplification bias.

（2）Multiplex PCR Library Preparation

A multiplex primer set covering the TCR V and J genes is used to directly amplify the rearranged CDR3 region (including part of the V region). After target amplification in the first round of PCR, the second round of PCR incorporates sequencing adapters and unique dual indices (UDI) to complete library construction; libraries are pooled in equal amounts following library QC and quantification.

（3）High-Throughput Sequencing and Data Analysis

Sequencing is performed on the Illumina platform to read out CDR3 sequences. Following quality filtering and sequence alignment (against the IMGT database), clonotype identification and quantification are completed, enabling systematic, multidimensional immune repertoire analysis.

3. Features and Advantages

· Broad sample compatibility: Supports gDNA, FFPE, and precious samples (DNA-based approach), with relatively relaxed sample quality requirements.

· Robust quantification: Under the DNA-based approach, each T cell carries one rearrangement template, enabling accurate estimation of clonal frequency on a per-cell basis.

· High sensitivity: Suitable for low-frequency clone detection and applicable to MRD-related research.

· Mature workflow: Aligned with the FDA-cleared MRD detection method (clonoSEQ), conferring high regulatory acceptance.

· Optional UMI (RNA-based approach): Further corrects for amplification bias and improves quantification precision.

· Favorable cost and turnaround: A standardized workflow that supports scalable operation.

Scope and limitations: This route focuses primarily on CDR3 rather than the full-length V(D)J sequence, and is a bulk sequencing approach (it does not provide single-cell–level α–β chain pairing). Differences in primer efficiency may introduce a degree of amplification bias. If full-length sequences, unbiased amplification, or native α–β pairing are required, we recommend TCR 5'RACE full-length sequencing or single-cell TCR immune repertoire sequencing.

4. Applications

· Hematologic malignancy clonality and MRD research: Clonality determination and dominant clone tracking (research use).

· Immune monitoring and clone tracking: Dynamic analysis of T cell clones before and after treatment and across different time points.

· FFPE retrospective studies: Cohort analysis using archived tissue samples.

· Cell therapy research: Robust quantification of T cell clonal dynamics, supporting the research characterization of products such as CAR-T.

5. Report and Deliverables

The report covers the complete content from sequencing quality control to systematic immune repertoire analysis. Core analysis modules include:

Deliverables: Illustrated analysis report (PDF) + detailed clonotype data table (Excel/CSV) + raw sequencing data (FASTQ)

Figure 2. Schematic of Multidimensional TCR Immune Repertoire Analysis Results

6. Service Workflow

7. Sample Requirements

*Note: Specific input amounts and shipping requirements are subject to the latest Sample Submission Form; consultation prior to sample submission is recommended to confirm the plan.

8. Technical Specifications

9. References

[1] U.S. Food and Drug Administration. Hematologic Malignancies: Regulatory Considerations for Use of Minimal Residual Disease in Development of Drug and Biological Products. 2020.

[2] U.S. Food and Drug Administration. Considerations for the Development of CAR T Cell Products; Draft Guidance. 2024.

[3] van Dongen JJ, et al. EuroClonality-NGS Working Group. Standardized next-generation sequencing of immunoglobulin and T-cell receptor gene recombinations. Leukemia. 2012.

[4] Center for Drug Evaluation, National Medical Products Administration. Technical Guidelines for Clinical Trials of Immune Cell Therapy Products (Trial). Announcement No. 15 of 2021.