Benchmarking Evercode TCR for Rare Clonotype Detection in Single Cell TCR Profiling

Key Takeaways:

• 0.1% detection sensitivity. Evercode TCR identified Jurkat-derived CDR3α/β sequences present in just 1 in 1,000 cells in a controlled spike-in experiment.
• Quantitative accuracy across titration. Observed Jurkat frequencies across the titration series (0.1–10 %) show high concordance with known inputs (R² > 0.99).
• Whole transcriptome context for every cell. Evercode TCR simultaneously profiles gene expression and paired CDR3α/β TCRs, enabling repertoire characterization of distinct T cell subtypes.

Experimental Design:

The immoralized human T cell line Jurkat E6-1 was selected as a reference standard due to its uniform T cell receptor (TCR) expression and previous use in TCR sequencing benchmarking experiments¹.

Jurkat E6-1 T cells and enriched CD3+ T cells from healthy donor peripheral blood mononuclear cells (PBMCs) were fixed using Evercode Fixation to stabilize the cell structure and protect the RNA transcriptome from degradation.

To assess the sensitivity of Evercode TCR, Jurkat E6-1 T cells were spiked into T cell-enriched PBMCs at defined ratios of 100%, 10%, 1%, and 0.1% (Figure 1). Samples were barcoded using Evercode TCR and sequenced on the Illumina Novaseq X.

Figure 1. Jurkat E6-1 T cells and CD3+ T cell-enriched PBMCs were mixed at four different percentages and barcoded using Evercode TCR.

Importantly, we validated Jurkat E6-1 cells as a reference standard in our experimental setup using single cell TCR profiling of a pure population of Jurkat E6-1 cells. For both the TCR α and β chains the characteristic Jurkat clonotype comprising CDR3α (CAVSDLEPNSSASKIIF) and CDR3β (CASSFSTCSANYGYTF) motifs were observed in ~97% of cells.

Figure 2. Jurkat cells exhibit a dominant, consistent clonotype. In pure samples, ~97% of cells expressed the expected CDR3α/β motifs, with a ~65% pairing rate.

Results:

Barcoded and sequenced cells (n = 110,067 T cells) were analyzed with the Parse Biosciences Analysis Pipeline v1.6. Among these, ~68% of cells were assigned productive TRA and TRB pairs (see Summary Report). UMAP clustering clearly resolved Jurkat cells from donor-derived T cells, with the Jurkat cluster correctly assigned based on their characteristic CDR3α/β sequences (Figure 3).

Figure 3. UMAP clustering reveals clear separation of Jurkat and PBMC donor-derived T cells. The Jurkat population is enriched for the known clonotype (purple) and PBMC donor-derived T cells do not exhibit the Jurkat clonotype.

Observed Jurkat clonotype frequencies across spike-in conditions closely matched the theoretical input ratios (Figure 4). The correlation across the full 0.1–100% range exceeded R² = 0.99, confirming the sensitivity and accuracy of Evercode TCR.

Figure 4. Observed Jurkat frequencies closely match expected proportions across all spike-in conditions (0.1-100%).

Why this Dataset Matters

Detecting rare clonotypes is critical for understanding immune responses to infection, cancer, and autoimmunity, but their low frequency makes accurate identification challenging. This dataset demonstrates that Evercode TCR can reliably detect TCR clonotypes at frequencies as low as 0.1%, or one cell in a thousand for a 4 sample/100,000 cell experiment.

Notably, as the number of cells in a sample increases, the lower limit of detection with Evercode TCR can improve even further—enabling even rarer clonotypes to be identified in larger-scale experiments. Given the vast diversity of the T cell receptor repertoire, clonotypes of therapeutic relevance are often present at low abundance. Evercode TCR overcomes traditional limitations with industry-leading sensitivity and paired whole transcriptome context, enabling researchers to:

• Confidently detect and quantify rare, disease-relevant clonotypes
• Resolve the full transcriptional state of T cells carrying specific TCRs
• Advance repertoire-based diagnostics, vaccine design, and T cell therapy development

This Jurkat spike-in titration provides a controlled benchmark for assay performance, supporting future applications in immune monitoring and discovery.

Next Steps

Compare Evercode TCR with Other Platforms

See how researchers in the Ellebrecht Lab at the University of Pennsylvania evaluated Evercode TCR alongside alternative TCR profiling technologies and identified nearly 5 times the number of unique clonotypes

• Read the Customer Comparison

Learn more about the Evercode TCR Workflow
Dive into assay details, learn about applications, and see more data in the technical brochure.

• Download the Brochure