By EVOBYTE Your partner in bioinformatics
Introduction
Every T and B cell carries a unique “barcode” in its antigen receptor. Sequencing these barcodes—called adaptive immune receptor repertoire sequencing, or AIRR‑seq—lets us read immune history and forecast immune behavior. Two receptors dominate the field: T‑cell receptors (TCRs) and B‑cell receptors (BCRs). Both are created by V(D)J recombination, which stitches Variable (V), Diversity (D), and Joining (J) gene segments into a novel sequence. The most informative stretch is the CDR3 region, where antigen specificity is concentrated. In practice, AIRR‑seq delivers “clonotypes” (groups of cells sharing the same receptor), diversity metrics, and sometimes paired chains, all of which fuel biomarker discovery, immuno‑oncology, and vaccine R&D. Community standards such as MiAIRR and the AIRR Data Commons are now central to making these data reusable and comparable across studies.
From short reads to long reads: how we sequence TCRs and BCRs
Most projects still rely on short‑read targeted amplicons with unique molecular identifiers (UMIs) to correct PCR/sequencing errors, or on single‑cell V(D)J assays that capture paired chains and gene expression. 10x Genomics’ 5′ immune profiling is the de facto single‑cell approach; it reconstructs full‑length V(D)J transcripts per cell and reports clonotypes while specifying read lengths and depth needed to span the V‑J junction.
Long‑read platforms (PacBio HiFi and Oxford Nanopore) are rising because they sequence complete receptors—retaining isotype information for BCRs and the exact arrangement of framework and CDR regions. Recent protocols like FLIRseq report accurate, quantitative full‑length IR profiling and highlight advantages over CDR3‑only short‑read views. Still, long‑reads demand careful error‑correction and thoughtful benchmarking before clinical use.
On the analysis side, open tools such as MiXCR and IgBLAST are workhorses. MiXCR aligns reads, assembles clonotypes by CDR3 or VDJRegion, performs barcode‑aware error correction for UMI/single‑cell data, and exports AIRR‑compliant tables. IgBLAST remains a standard for V, D, and J gene assignment and CDR delineation against curated germline databases.
Bioinformatics challenges that make or break AIRR studies
AIRR‑seq is not “just sequencing.” Three pitfalls show up repeatedly:
- Error and bias: PCR jackpotting and sequencing errors inflate rare clonotypes. UMIs and quality‑guided clustering help, but only if your pipeline uses them correctly. MiXCR’s multi‑layer correction is one practical safeguard.
- Germline ambiguity: Novel or poorly cataloged germline alleles can mislead V/D/J calls and SHM rates. IgBLAST and AIRR standards evolve to improve allele coverage and reporting, but documenting metadata via MiAIRR remains essential for reproducibility.
- Clonotype definition and pairing: In bulk data, defining a clone by CDR3 alone can under‑ or over‑split true lineages, especially for hypermutated BCRs. Single‑cell V(D)J recovers true chain pairing and mitigates this, but requires adequate read length and depth across the V‑J junction to avoid dropouts.
For downstream exploration, R ecosystems like scRepertoire/immunarch integrate V(D)J with single‑cell expression, enabling clonality and diversity comparisons across conditions.
Key terms you’ll see in methods and reports—and why they matter:
– CDR3: primary specificity hotspot; anchors clonotype definitions.
– UMI: tracks unique molecules to correct amplification bias.
– SHM (somatic hypermutation): BCR‑specific edits that map affinity maturation; crucial in vaccine studies.
– Clonotype: the analytical unit linking sequence to cell expansion.
– AIRR/MiAIRR: data and metadata standards that make results comparable and shareable.
Why this matters for immuno‑oncology, biomarkers, and vaccines
In oncology, TCR/BCR repertoire features are being tested as predictive biomarkers and for pharmacodynamic readouts under checkpoint blockade. Single‑cell V(D)J plus RNA profiles can track intratumoral clonal expansion and phenotype shifts, supporting mechanism‑of‑action studies and combination strategies.
In hematologic malignancies, clinical assays already leverage receptor sequencing to quantify measurable residual disease (MRD). The FDA’s de novo authorization of clonoSEQ underscored the role of NGS‑based receptor tracking in guiding care decisions by detecting one cancer cell among a million. That decision paved the way for broader MRD applications and set performance expectations for similar tests.
For vaccines, repertoire sequencing reveals how BCR lineages expand and mature after prime and boost, helping teams compare platforms, select adjuvants, and refine immunogen design. Emerging long‑read methods add isotype and full‑length context, improving insights into class switching and mutation trajectories that short‑reads might miss.
Summary / Takeaways
AIRR‑seq turns immune receptors into analyzable data—clonotypes, diversity, and chain pairing—that accelerate biomarker discovery and mechanism‑driven therapeutics. Today’s pragmatic stack combines UMI‑aware targeted amplicons, single‑cell V(D)J for pairing, and increasingly, long‑reads for full‑length context. Lean on community standards (MiAIRR, AIRR Data Commons) and validated tools (MiXCR, IgBLAST) to keep analyses robust and comparable. If you’re starting a project this quarter, define clonotype rules up front, budget depth to span the V‑J junction, and plan for metadata that meets MiAIRR—your future self (and reviewers) will thank you.