BCR Multiplex PCR Sequencing

BCR Multiplex PCR Sequencing

1. Background

The clonal diversity of the B cell receptor (BCR) and its secreted form—the antibody—lies at the core of humoral immune responses. The BCR is composed of a heavy chain (IgH) and a light chain (κ or λ), and its variable region continuously evolves through V(D)J rearrangement and somatic hypermutation (SHM), giving rise to a highly individualized immune repertoire. Resolving the clonal composition and dynamic changes of the BCR is of major significance for B cell tumor clonality assessment, minimal residual disease (MRD) monitoring, and the early-stage research of antibody therapeutics. Using a primer set covering the immunoglobulin V and J genes, the multiplex PCR approach directly amplifies the IgH and light-chain rearrangement regions, is compatible with both gDNA and FFPE, and represents the technical route with the broadest sample compatibility and most robust quantification among comparable methods.

This route has clear regulatory precedent: clonoSEQ (Adaptive Biotechnologies)—the first and currently the only FDA-cleared (De Novo, DEN170080) NGS-based in vitro diagnostic for MRD—is built precisely on multiplex PCR + NGS with gDNA-based detection of immunoglobulin gene rearrangements as its core technology. It is indicated for MRD quantification in multiple myeloma, B-ALL, and CLL, and has been incorporated into the NCCN guidelines. The NGS standardization framework for IG rearrangements established by the EuroClonality-NGS consortium on the basis of the BIOMED-2 primer system has further cemented this route's international standard-setting status in clonality determination and MRD research.

ZhuHai provides end-to-end services spanning sample QC, multiplex PCR library preparation, high-throughput sequencing, and systematic bioinformatic analysis, helping clients precisely resolve B cell clonal dynamics in a mature and cost-effective manner—delivering a high-quality, traceable data foundation for clonality determination, MRD monitoring, and antibody-related research.

2. Technical Principle

Figure 1. Workflow of Multiplex PCR Amplification of the Immunoglobulin 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 can be combined with constant-region primers to enable easier isotype discrimination.

（2）Multiplex PCR Library Preparation

A multiplex primer set covering the IgH (as well as κ/λ light-chain) V/J genes (following the BIOMED-2/EuroClonality-NGS primer strategy) is used to directly amplify the rearranged CDR3 region (including part of the V region, e.g., the FR1/FR2/FR3 segments). 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); one of the most broadly compatible routes among comparable methods.

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

· Regulatory maturity: Shares the same technical route as the FDA-cleared clonoSEQ (DEN170080)—multiplex PCR + NGS, gDNA-based—conferring high regulatory acceptance.

· High sensitivity: Suitable for B cell clonality determination and MRD-related research.

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

Scope and limitations: Differences in primer efficiency may introduce a degree of amplification bias; B cell somatic hypermutation (SHM) may occur at V-region primer binding sites, reducing the amplification efficiency of some clones—an inherent limitation of BCR multiplex PCR. Isotype information is difficult to obtain with the DNA-based approach (the RNA-based approach with constant-region primers enables easier discrimination). This route focuses primarily on CDR3 rather than the full-length sequence, and is a bulk sequencing approach (it does not provide VH–VL pairing). If full-length sequences, isotype information, SHM analysis, or heavy-/light-chain pairing are required, we recommend BCR 5'RACE full-length sequencing or single-cell BCR immune repertoire sequencing.

4. Applications

· B cell tumor clonality and MRD research: Clonality determination and dominant clone tracking (research use).

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

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

· Antibody drug development support: Resolving the amplification patterns of B cell clones to support early-stage research for antibody sequence discovery.

Figure 2. Schematic of BCR Clonality Assessment and MRD Dynamic Monitoring

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).

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] Adaptive Biotechnologies. clonoSEQ Assay — FDA De Novo Authorization DEN170080. 2018.

[3] van Dongen JJ, et al. EuroClonality-NGS Working Group. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations. Leukemia. 2003.

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