SMARTer Human TCR a/b Profiling Kit v2—flexible kit with ultimate sensitivity for understanding the TCR repertoire
The SMARTer Human TCR a/b Profiling Kit v2 (TCRv2) is powered by robust chemistry that provides unparalleled sensitivity and reproducibility. The kit leverages SMART (Switching Mechanism at 5' end of RNA Template) full-length cDNA synthesis technology and pairs NGS with a 5'-RACE approach to capture the complete V(D)J variable regions of TRA and TRB genes.
The SMARTer Human TCR a/b Profiling Kit v2 (TCRv2) is powered by robust chemistry that provides unparalleled sensitivity and reproducibility. The kit leverages SMART (Switching Mechanism at 5' end of RNA Template) full-length cDNA synthesis technology and pairs NGS with a 5'-RACE approach to capture the complete V(D)J variable regions of TRA and TRB genes.
The kit is designed to work with a range of RNA input amounts (RIN ≥8; depending on the sample type) and has been shown to yield high-quality sequencing libraries from as little as 10 ng to 1 µg of total RNA obtained from peripheral blood leukocytes, 1 ng to 100 ng of total RNA obtained from T cells, or from 1,000 to 10,000 purified, whole T cells. Libraries can be generated to obtain both alpha and beta chain diversity information. This latest profiling kit also includes unique molecular identifiers (UMI), making it possible to remove reads derived from PCR duplicates and sequencing errors, thus ensuring more accurate and reliable results. In addition to the reagents, the TCRv2 kit includes a dual index (UDI) plate to generate Illumina-compatible sequencing libraries.
Once output files are generated with an Illumina sequencer, they can be analyzed using the Cogent NGS Immune Profiler Software. This software enables high-quality TCR profiling analysis, including count and identification of clonotypes and sequence information for V(D)J regions.
Benefits of this kit include:
- Use of UMIs to correct for PCR and sequencing errors
- Addition of UDIs, allowing for greater confidence in sequencing on a patterned flow cell (such as the NovaSeq™ system) and the ability to pool a greater number of samples
- Flexibility to sequence on any Illumina instrument
- Full-length V(D)J analysis on the MiSeq™ system
- Access to Cogent NGS Immune Profiler Software, an easy-to-use analysis pipeline tool for users of any bioinformatic experience level, for faster data analysis
Overview
- Compatible with a wide range of sample inputs: total RNA (10 ng to 1 µg from peripheral blood leukocytes or 1 ng to 100 ng from T cells) or purified, whole T cells (1,000 to 10,000 cells)
- Simple PCR amplification: a single primer pair for each TCR (alpha or beta) subunit per reaction
- UMI-based correction: removal of reads derived from PCR duplicates and sequencing errors
- Sensitive and specific clonotype detection: optimized cDNA library generation
- UDI implementation: increased multiplexing and confidence for sequencing on high-throughput sequencers
- Illumina-ready sequencing libraries: Illumina-compatible index sequences are incorporated for multiplexing up to 192 libraries in a single run
- Flexible sequencing options: either full-length V(D)J information on the MiSeq™ system or CDR3 information on all Illumina platforms
- Complete workflow: robust chemistry complemented with use of our Cogent NGS Immune Profiler Software* for sequencing data analysis
*The Cogent NGS Immune Profiler Software is free to use with this kit for both academic and for-profit entities
Figure 1. Sensitive and reproducible clonotype detection from a broad range of RNA amounts. TRA and TRB libraries were generated from 1, 10, and 100 ng of human CD3+ T-cell total RNA and 1,000 and 10,000 CD3+ T cells. The sequence reads were processed by the Cogent NGS Immune Profiler Software.
| % Jurkat RNA spiked in to 100 ng of PBMC RNA | Total read count (TRA/TRB) | Without UMI collapse | With UMI collapse | ||||
|---|---|---|---|---|---|---|---|
| # of TRB raw reads | # of reads for TRBV12‑3-TRBJ1‑2 | Detected percentage of Jurkat reads | # of detected UMIs | # of UMIs for TRBV12‑3-TRBJ1‑2 | Detected percentage of Jurkat UMIs | ||
| 10.0% | 2,500,000 | 1,565,005 | 397,179 | 25.0% | 281,280 | 62,629 | 22.0% |
| 1.0% | 2,500,000 | 1,422,102 | 47,160 | 3.3% | 219,776 | 6,426 | 2.9% |
| 0.1% | 2,500,000 | 1,366,127 | 5,412 | 0.4% | 189,580 | 631 | 0.33% |
| 0.01% | 2,500,000 | 1,218,025 | 521 | 0.043% | 196,615 | 74 | 0.038% |
| 0.001% | 2,500,000 | 1,331,465 | 909 | 0.068% | 197,870 | 6 | 0.003% |
| 0.0001% | 2,500,000 | 1,409,199 | - | 0% | 124,149 | - | 0% |
| 0% | 2,500,000 | 1,222,245 | - | 0% | 197,933 | - | 0% |
Table 1. Assessing the sensitivity and reproducibility of the SMARTer approach. Spike-in analysis was performed in replicate on PBMC RNA samples spiked at varying concentrations (10%, 1%, 0.1%, 0.01%, 0.001%, and 0.0001%) with RNA obtained from a homogeneous population of leukemic Jurkat T cells (containing TRBV12-3-TRBJ1-2 clonotypes). TRB CDR3 regions were amplified from 100 ng of total RNA using the TCRv2 kit and sequenced. Reads of 2 x 150 bp were obtained on an Illumina NextSeq® system. The sequencing reads were downsampled to 2.5 M reads. Read results for spike-in concentrations identified as the reliable concentration limit for each criterion (without and with UMI collapse) have data highlighted in gray. Without UMI collapse, PCR duplicates of TRBV12-3 were observed in 0.0010% of the raw reads.
Figure 2. Superior sensitivity and reproducibility with TCRv2. We split 5M PBMC cells from two different healthy donors for RNA and gDNA extraction. 1.6 µg of gDNA was used for library preparation according to the manufacturer's instructions (15% of the total amount of extracted gDNA). 100 ng of RNA was used for library preparation (2% of the total amount of extracted RNA). Clonotype numbers for TCRa/b libraries are shown from each company (NT: not tested). In the comparison, TCRv2 generated an average of 48.7K and 163K clonotypes for TRA and TRB, respectively, representing a 290% increase against Company X's RNA-based approach and a 145% increase against Company Y’s gDNA-based approach. Importantly, the RNA methods used only 2% of the total RNA from the 5M PBMCs.
More Information
Applications
Human TCR repertoire analysis (TCRα and TCRβ subunits)
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