1. DriverMap™ AIR DNA Profiling Assay

The DriverMap™ AIR TCR/BCR DNA Profiling Kits combine multiplexed PCR amplification with the depth and precision of Next-Generation Sequencing (NGS) to quantitatively measure TCR or BCR rearranged gene copy number (corresponding to a number of T and B cells) and analyze the CDR3 sequences of immune receptor genes. AIR TCR and AIR BCR Kits allow simultaneous profiling of CDR3 repertoire for all four TCR chains (TRA, TRB, TRD, TRG) and all three BCR chains (IGH, IGK, and IGL), respectively, in separate reactions.

Main advantages of DriverMap™ AIR DNA Assay:

• An easy-to-run, one-tube, single-day assay without intermediate purification step allows unbiased, comprehensive profiling of CDR3 regions for all seven TCR/BCR chains from genomic DNA isolated from whole blood, PBMC, tissue, FFPE or directly from a small number (e.g., 100-50,000) of purified immune cells without prior DNA isolation.
• The assay provides robust, quantitative, and reproducible measurements of the DNA clonotype copy number over as much as 4-orders of magnitude differences in representation level.
• The DriverMap protocol employs several mechanisms to minimize the amplification of off-target and primer dimer background products.
• A redundant set of experimentally validated forward primers (designed for variable FR3 region) and reverse primers (designed for the J region) are designed for functional TCR and BCR genes with the exclusion of non-functional pseudogenes and ORFs (as defined by IMGT database https://www.imgt.org/IMGTrepertoire/).
• The indexing primers are designed with Illumina’s DNA 10-n UD Indexes to minimize NGS index-swapping background issues, and amplified products are compatible with 300-n paired-end NGS sequencing using Illumina’s NextSeq/NovaSeq instruments.
• The reverse gene-specific primers are designed with Unique Molecular Identifiers (UMIs) to facilitate accurate quantification of the copy number of TCR/BCR DNA molecules used in amplification steps and detection of low abundance clonotypes above background level.
• The concentration of reverse gene-specific primers is optimized to provide balanced amplification efficiency of all chains for TCR (TRA, TRB, TRD, and TRG) or BCR (IGH, IGK, and IGL) reactions.
• NGS data can be analyzed with the MiXCR software package available online. Cellecta also provides custom MiXCR data analysis services on request.

Fig.1. Outline of AIR DNA multiplex-PCR technology. Step1: Reverse gene-specific primers (GSP) with UMI targeting TCR or BCR J-region isoforms (in separate reactions) are annealed to single, complementary DNA strand (top strand) and extended with DNA polymerase. Step 2: Forward GSPs are annealed to extended DNA template and after extension by DNA polymerase generate CDR3 regions flanking from both sides by universal anchor sequences (AP1 and AP2). Step 3: In the first PCR step, anchored universal primers (AP1 and AP2) amplify CDR3 DNA fragments. Step 4: The second PCR step amplifies CDR3 fragments using indexed primers. The indexed amplified products (separately for TCR and BCR) are combined in equal amounts and analyzed by NGS.

Additional Profiling Assays Compatible with the AIR DNA Assay:

• DriverMap™ AIR TCR-BCR Profiling Kit (human RNA) is designed to generate comprehensive AIR CDR3 repertoire profiling of all seven TCR (TRA, TRB, TRD, and TRG) and BCR (IGH, IGK, and IGL) chains from RNA samples. AIR RNA assay provides higher sensitivity in detecting CDR3 clonotypes than AIR DNA assay, especially from small biological samples. Both AIR RNA and AIR DNA assay could be effectively used together to identify antigen-induced clonotypes. For more details, please refer to AIR Technical Guide.
• DriverMap™ T/B Immune Marker Profiling Kit is designed for deep characterization of T and B immune cell fractions (e.g., naïve, effector, memory, exhausted, etc.) using a set of primers designed for 500 highly informative T-cell and B-cell subtyping and activation marker genes. Combined profiling of AIR repertoire and T/B marker genes in isolated (e.g., by FACS or magnetic beads) immune cell fractions provide a comprehensive phenotypic characterization of T/B cell subtypes associated with different TCR/BCR clonotypes.
• DriverMap™ EXP Genome-Wide Profiling Kit is a convenient single-tube multiplex RT-PCR-NGS protocol that robustly measures the expression level of all 19,000 human protein-coding genes starting from a small amount of total RNA (down to single-cell level). Combined EXP and AIR profiling can be used for comprehensive phenotypic characterization of RNA samples and unbiased discovery of novel biomarkers associated with specific CDR3 clonotypes.

Related Products and Services:
For more detailed recommendations for your experimental design, please refer to the AIR Technical Guide. For more information on Cellecta’s DriverMap kits and services, please visit the DriverMap Adaptive Immune Receptor (AIR) Profiling Service webpage.

Please read the entire user manual before proceeding with your experiment.

*Click the ⤓ Download as PDF link at the bottom of the left menu to download the PDF version of this user manual.

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