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Overview
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Overview

Analyzing paired chain information of T cell receptors (TCR) and B cell receptors (BCR) at the single-cell level is a powerful tool for probing T and B cell diversity and function in lymphoid malignancies, infectious diseases, autoimmune disorders and tumor immunology. The BD Biosciences TCR and BCR profiling tools help you uncover clonal diversity and function of T and B cells, respectively, at the single-cell level.

 

BD Rhapsody™ TCR/BCR Multiomic Assay

The BD Rhapsody™ TCR/BCR Multiomic Assay helps you assemble the full length VDJ sequence for the T cell and B cell receptor while assessing transcriptomic and protein expression of the same cells. The assay can serve as the one-stop shop for your single-cell multiomics needs.

 

The BD Rhapsody™ TCR/BCR Multiomic Assay includes:

  • Full multiomics optimization and validation
  • Reagents and pipeline to support assembly of full-length TCR and BCR sequences
  • Compatibility with whole transcriptome and targeted mRNA assays
  • Enhanced cell capture beads for sensitive molecule detection

 

BD Rhapsody™ VDJ CDR3 Protocol with V1 beads

The BD Rhapsody™ VDJ CDR3 Protocol utilizes the existing BD Rhapsody™ Targeted Kits and the human/mouse immune response primer panel and is designed to work alongside the BD® AbSeq Assay and BD® Single-Cell Multiplexing Kit (SMK). Details about the individual reagents needed to run the assay are included in the respective protocols. A dedicated bioinformatics pipeline is also available for you to analyze sequencing data generated using the CDR3 protocol.

 

The BD Rhapsody™ VDJ CDR3 Assay for profiling TCR and BCR includes:

  • Validated primer sequences
  • A detailed step-by-step protocol for analyzing the human or mouse CDR3 region of TCR and BCR using the BD Rhapsody™ Single-Cell Analysis System

 

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Features

The BD Rhapsody™ TCR/BCR Multiomic Assay has been fully validated with our new enhanced cell capture beads as well as BD® AbSeq Antibody-Oligos and the BD® Single-Cell Multiplexing Kit (SMK). Confidently analyze the transcriptome, proteome and full length TCR/BCR repertoire of your cells, including gamma/delta T cells, from the only single-source provider of single-cell multiomics products.

 
BD Rhapsody TCR/BCR Multiomic Assay Features
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The BD Rhapsody™ TCR/BCR Multiomic Assay can be run with an mRNA assay alone without any antibody staining or can be utilized with different combinations.

Applications

A single workflow to profile the T cell and B cell receptors validated with existing targeted or WTA mRNA assays as well as protein analysis and sample multiplexing

 

In the experiment outlined below, resting human PBMCs, stained with the BD® AbSeq Immune Discovery Panel and four drop-in BD® AbSeq Antibodies, were multiplexed with enriched T cells stained with a 15-plex AbSeq panel. The pooled samples were processed with the TCR/BCR multiomic assay and WTA assay (Cat. No. 633801). The following example analysis was conducted on the T cells within the PBMC data. 

Step 1:

 Using both protein and gene expression a user can generate a tSNE plot and use AbSeq to identify cell populations.

 

tSNE plot (right) with major cell lineages identified. CD8 positive cells are highlighted and analyzed deeper as shown in subsequent figures.

 
Step 1
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Step 2:

An unsupervised phenograph analysis can be performed for a population of interest

 

Six distinct CD8 positive subpopulations (right) were identified through an unsupervised phenograph analysis.

 
Step 2
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Step 3:

Frequencies of unique clonotypes within clusters can be reported

 

Expanded paired TCR clonotypes for alpha and beta chains (right) were identified within phenograph 2 of CD8 positive cells.

 
Step 3
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Step 4:

Cells that have the most frequent clonotype can be identified on tSNE plot.

 

The location of individual cells harboring the expanded paired clonotypes were identified by overlaying data on the tSNE plot (right).

 
Step 4
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Step 5:

Using AbSeq and WTA information, differential gene and protein expression analysis of cells with the most frequent clonotype vs the rest of the population can be performed

 

Gene and protein expression data for cells with the most frequent clonotype are compared to remaining CD8 positive cells (right).

 
Step 5
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Step 6:

Cell IDs and chain information can be obtained from pipeline output files

 

The cell label and CDR3 information for cells expressing the expanded clonotype were identified and used for further analysis.

 
Cell_Index Chain_Type CDR3_Translation_Dominant
54044 TCR_Alpha AVLAEGGKLI
54044 TCR_Beta ASSLWGSPLH
123296 TCR_Alpha AVLAEGGKLI
123296 TCR_Beta ASSLWGSPLH
123563 TCR_Alpha AVLAEGGKLI
123563 TCR_Beta ASSLWGSPLH
147961 TCR_Alpha AVLAEGGKLI
147961 TCR_Beta ASSLWGSPLH

Step 7:

VDJ and C gene information can be determined for clonotype of interest

 

The translated V(D)J gene segments for cells' labels of interest were tabulated from bioinformatic ouptut files.

 

 

Cell_Index Chain_Type V_gene_Dominant D_gene_Dominant J_gene_Dominant C_gene_Dominant Full_Length Productive
54044 TCR_Alpha TRAV20*01   TRAJ23*01 TRAC TRUE TRUE
54044 TCR_Beta TRAV27*01 TRBD1*01 TRBJ1-6*02 TRBC1 TRUE TRUE
123296 TCR_Alpha TRAV20*01   TRAJ23*01 TRAC TRUE TRUE
123296 TCR_Beta TRAV27*01 TRBD1*01 TRBJ1-6*02 TRBC1 TRUE TRUE
123563 TCR_Alpha TRAV20*01   TRAJ23*01 TRAC TRUE TRUE
123563 TCR_Beta TRAV27*01 TRBD1*01 TRBJ1-6*02 TRBC1 TRUE TRUE
147961 TCR_Alpha TRAV20*01   TRAJ23*01 TRAC TRUE TRUE
147961 TCR_Beta TRAV27*01 TRBD1*01 TRBJ1-6*02 TRBC1 TRUE TRUE

Step 8:

Full length sequence of clonotype can be obtained (nucleotide sequence also available)

 

 

Cell_Index VDG_Translation_Trimmed
54044 EDQVTQSPEALRLQEGESSSLNCSYTVSGLRGLFWYRQDPGKGPEFLFTLYSAGEEKEKERLKATLTKKESFLHITAPKPEDSATYLCAVLAEGGKLIFGQGTELSVKP
54044 EAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSLWGSPLHFGNGTRLTVT
123296 EDQVTQSPEALRLQEGESSSLNCSYTVSGLRGLFWYRQDPGKGPEFLFTLYSAGEEKEKERLKATLTKKESFLHITAPKPEDSATYLCAVLAEGGKLIFGQGTELSVKP
123296 EAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSLWGSPLHFGNGTRLTVT
123563 EDQVTQSPEALRLQEGESSSLNCSYTVSGLRGLFWYRQDPGKGPEFLFTLYSAGEEKEKERLKATLTKKESFLHITAPKPEDSATYLCAVLAEGGKLIFGQGTELSVKP
123563 EAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSLWGSPLHFGNGTRLTVT
147961 EDQVTQSPEALRLQEGESSSLNCSYTVSGLRGLFWYRQDPGKGPEFLFTLYSAGEEKEKERLKATLTKKESFLHITAPKPEDSATYLCAVLAEGGKLIFGQGTELSVKP
147961 EAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSLWGSPLHFGNGTRLTVT

 

 

The full-length amino acid sequence of the V(D)J alpha and beta TCR chains shown here were annotated showing framework and CDR regions in different colors.

 FR1  CDR1  FR2  CDR2  FR3  CDR3  FR4
TCR_alpha EDQVTQSPEALRLQEGESSSLNCSYTVSGLRGLFWYRQDPGKGPEFLFTLYSAGEE KEKERLKATLTKKESFLHITAPKPEDSATYLCAVLAEGGKLIFGQGTELSVKP
TCR_beta EAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSLWGSPLHFGNGTRLTVT
BD Rhapsody™ VDJ CDR3 Assay
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FEATURES

The BD Rhapsody™ VDJ CDR3 Assay can be used with the BD Rhapsody™ Targeted Kit, BD® AbSeq Assay and BD® Single-Cell Multiplexing Kit, providing flexibility in your research

 

Based on the research need, the assay can be used in any of the following combinations:

 

  1. BD Rhapsody™ VDJ CDR3 Protocol—utilizes the BD Rhapsody™ Targeted Kit + Human Immune Response Panel* and enables you to analyze TCR and BCR sequences.
  2. BD Rhapsody™ VDJ CDR3 + SMK Protocol—utilizes the BD Rhapsody™ Targeted Kit + Human Immune Response Panel* + SMK and enables you to process multiple samples together in a single cartridge.
  3. BD Rhapsody™ VDJ CDR3 + BD® AbSeq Protocol—utilizes the BD Rhapsody™ Targeted Kit + Human Immune Response Panel* + BD® AbSeq Assay and enables multiomic analyses integrating TCR and BCR sequence analyses alongside protein information on the same sample.
  4. BD Rhapsody™ VDJ CDR3 + BD® AbSeq + SMK Protocol—requires the BD Rhapsody™ Targeted Kit + Human Immune Response Panel* + BD® AbSeq Assay + SMK and enables you to perform multiomic analyses on multiple samples of interest together in a single cartridge.

 

*While the VDJ protocol can technically work with any targeted panel, it works best with the immune response panel. To work effectively with our CDR3 VDJ assay, we recommend including immune response panel makers in custom panels.

PERFORMANCE

A powerful assay for analyzing CDR3 information of TCR and BCR

The hallmark of the BD Rhapsody™ VDJ CDR3 Protocol is its high sensitivity and specificity in detecting distinct clonotypes of T and B cells including gamma delta T cells alongside protein analyses.

APPLICATIONS

A multiomic tool for performing in-depth immune cell profiling

   

    

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