TIRTL-seq is a T cell receptor (TCR) sequencing technique, presented in the paper “TIRTL-seq: deep, quantitative and affordable paired TCR repertoire sequencing” by researchers from St. Jude Children’s Research Hospital in Nature Methods. The researchers present a universal, high-throughput and rapid TCR library sequencing protocol that offers significant improvements over previous techniques.
Every adaptive immune response tells a story which begins with the T cell. Understanding how T cells recognize disease hinges on a deceptively simple detail: the amino acid sequences and exact pairing of the T-cell receptor’s two chains, α and β. This pair acts as a molecular sensor that allows T cells to detect pathogens, cancer cells, and other immune challenges. Yet capturing this pairing at scale has remained one of immunology’s biggest technical hurdles.
For researchers trying to understand immune responses across large patient cohorts, or for biotech teams developing next-generation immunotherapies, the field has been stuck choosing between depth, accuracy, and affordability.
We spoke with Dr. Mikhail Pogorelyy, a computational immunologist and co-developer of TIRTL-seq formerly at St. Jude, and now at Fred Hutch Cancer Center.
“There was no method able to do deep paired TCR repertoire sequencing for relatively cheap. That is what we wanted to solve.”
The researchers used the I.DOT Non Contact Dispenser to scale TIRTL-seq to 384 wells. The use of the I.DOT was critical to minimize risk of contamination.
Each T-cell receptor is a heterodimer: one α chain, one β chain, each encoded by independently rearranged gene segments. Bulk TCR sequencing breaks cells open and mixes all transcripts together, destroying the ability to know which α-β pair belongs to which T cell.
“If you lyse the cells, all the transcripts get mixed up. You don’t know which TCR-alpha is paired to which TCR-beta,” Dr. Pogorelyy explains. “Single-cell methods solve that, but they are too low-throughput and too expensive to do at the scale the field needs.”
He further spoke of a clever idea from 2015 (Howie et al., 2015), which hinted at a solution: split the sample into many wells, sequence each one separately, and computationally infer pairings by overlapping well “co-occurrence” patterns. But early versions were costly, required RNA extraction, used large reaction volumes, and depended on proprietary components and code. Together, this meant almost no labs could adopt the technique.
TIRTL-seq (“T-cell Immune Repertoire Throughput and Lineage sequencing”) builds on the combinatorial idea but resolves the limitations that prevented earlier versions from becoming practical. The innovations fall into four key areas:
Adapted from Fig 1a in Pogorelyy et al (2025): In brief, a cell suspension is distributed into 384-well plates containing an RT–lysis master mix under a hydrophobic overlay using noncontact liquid dispensers. After the RT reaction, PCR I master mix with V-segment and C-segment primers is dispensed into the same plate. The PCR I product is then diluted and transferred to the PCR II plate for indexing PCR with well-specific unique dual indices. The PCR II products are pooled by centrifugation, purified, size-selected using magnetic beads and sequenced on an Illumina platform. Total library preparation cost is listed for one 384-well plate.
The performance reported in the Nature Methods study marks a major leap in TCR sequencing:
“We applied it to really massive human peripheral blood samples to get up to the million of unique paired receptors out, as a proof of concept,” Pogorelyy notes.
These numbers shift TCR repertoire analysis from being a specialized capability available only to the best-resourced labs to something far more democratized and practical.
Scientific reproducibility has been a growing concern across fields of research. The TIRTL-seq team committed early to ensuring that other labs could implement, and trust, the method.
“We tried to be really reproducible,” commented Pogorelyy. “There’s a detailed protocol, all the data, all the software. And we even made a fully manual version so labs can try the method before committing to automation.”
This includes:
This hybrid approach, manual for accessibility and automated for throughput plus miniaturization, helps ensure the method reaches as many labs as possible.
For immunotherapies, the ability to discover rare tumor-reactive TCRs is a fundamental bottleneck. TIRTL-seq directly addresses that gap.
“If you can find TCRs recognizing a tumor, you can engineer patient cells to express them,” Dr. Pogorelyy explains. “This method finally lets us look at millions of T cells and see paired receptors to track immune responses or develop new therapies.”
Broader applications of TIRTL-seq include:
As the technology matures, TIRTL-seq could become a routine tool in both academic and industrial immune-monitoring workflows.
TIRTL-seq brings the field closer to a long-awaited ideal: high-resolution, affordable, large-scale paired TCR sequencing. For biotech companies, translational immunologists, and researchers studying the complexities of immune responses, it opens the door to deeper, broader, and more routine repertoire profiling.
As Pogorelyy puts it, “This technology was missing before. Now we finally have the ability to sequence T-cell receptors really deeply, and affordably!”
The researchers chose the I.DOT as their liquid handler of choice. The I.DOT non-contact dispenser enables effortless nanoliter dispensing, ensuring precise reagent delivery with exceptional accuracy and reproducibility. Together with the disposable source wells, the authors could feel safe that they eliminated any risk of cross-contamination.
Ready to experience the I.DOT advantage? Download the I.DOT brochure and learn how the I.DOT Non-Contact Dispenser can revolutionize your single-cell research.
Pogorelyy, M.V., Kirk, A.M., Adhikari, S. et al. TIRTL-seq: deep, quantitative and affordable paired TCR repertoire sequencing. Nat Methods (2025). https://doi.org/10.1038/s41592-025-02907-9