KIR/HLA Genotyping (Long-Read NGS)

KIR Genotyping by Long-Read Sequencing

High-resolution donor typing and starting-material confirmation for allogeneic / off-the-shelf CAR-NK and cell-therapy products. Using single-molecule long-read sequencing (PacBio HiFi / Oxford Nanopore), the complete sequences of KIR genes are read directly, spanning highly homologous paralogs and structural variation to obtain allele-level genotypes and complete centromeric/telomeric haplotypes, together with the B-content score and KIR–HLA ligand pairing — providing reference-grade immunogenetic evidence for Master Cell Bank (MCB) / starting-material characterization, confirmation of NGS results, and IND/BLA regulatory submissions.

1. Background

Natural killer (NK) cells recognize HLA class I through killer-cell immunoglobulin-like receptors (KIR) and other receptors, and decide whether to kill based on the net result of inhibitory and activating signals. In allogeneic / off-the-shelf CAR-NK and cell therapy, donor KIR and HLA genotypes jointly determine NK alloreactivity and education, and are core variables for donor selection and immunogenetic characterization. The KIR locus at chromosome 19q13.4 is among the most structurally complex and polymorphic regions of the human genome, with great variation in gene number and structure across haplotypes.

Clinical evidence has translated this biology into practice: in haploidentical transplantation, a KIR-ligand mismatch is associated with reduced leukemia relapse (Ruggeri et al., 2002); selecting unrelated donors by the B-content score improves AML transplant outcomes in a disease-specific manner (Cooley et al., 2010); and cord-blood-derived CAR-NK has shown encouraging response rates and a favorable safety profile in early-phase trials (Liu et al., 2020). At the same time, KIR (chromosome 19) and HLA class I (chromosome 6) are independent and unlinked, so KIR must be interpreted together with HLA; these associations are correlative, disease-specific evidence and do not constitute a guarantee of product efficacy.

KIR is one of the most difficult regions of the human genome to genotype precisely: the gene family is highly homologous and copy-number variable, with structural variants such as deletions, duplications and fusions (e.g., KIR3DL1/2v). Short-read sequencing struggles to assign reads correctly among highly similar paralogs and cannot establish phase over long distances, leaving ambiguity for KIR2DL5A/B, KIR2DS1 versus KIR2DL1, KIR3DL1 versus KIR3DS1, and for assembling centromeric/telomeric haplotypes. Single-molecule long reads span entire gene regions, resolving paralog assignment, revealing structural variation and reconstructing phase directly: a long-range PCR-based Oxford Nanopore approach covers all KIR genes except KIR3DP1, with allele-level concordance of 97.2–99.5% against published data and >99.7% gene-content concordance with PCR-SSP, while resolving phase ambiguity (Downing et al., 2025; Downing and D'Orsogna, 2022).

From a regulatory standpoint, KIR/HLA genotyping is not a mandatory release attribute; it is a scientific input for donor selection and for immunogenetic characterization of the starting material / Master Cell Bank. Long-read sequencing provides reference-grade allele and haplotype evidence, suited to confirmation and high-stringency use cases. This service can serve as supporting evidence within the frameworks of the FDA guidance “Considerations for the Development of Chimeric Antigen Receptor (CAR) T Cell Products” (2024), ICH Q5D (characterization of cell substrates), ICH Q2(R2) (validation of analytical procedures), and China's NMPA (2017) and CDE (2022) guidelines. The method can be qualified fit-for-purpose or fully validated per ICH Q2(R2) according to the development stage.

2. Technical Principle

This service is built on single-molecule long-read sequencing (PacBio HiFi / Oxford Nanopore) that reads complete KIR gene sequences directly, using IPD-KIR/IPD-HLA as the reference allele databases, to span homologous paralogs and structural variation and obtain allele-level genotypes with complete centromeric/telomeric haplotypes. The core steps are as follows:

(1) Sample receipt and QC

High-molecular-weight genomic DNA (or donor-derived cells) is received; because long reads are more demanding on DNA integrity, fragment size and degradation are assessed carefully.

(2) Library preparation

Two routes are available: (i) long-range PCR amplifying all KIR genes except KIR3DP1, followed by library preparation (Nanopore route); or (ii) long-read library preparation with optional targeted enrichment (PacBio HiFi route).

(3) Long-read sequencing

PacBio HiFi obtains high per-base-accuracy long reads via circular consensus sequencing (CCS), or sequencing is performed by Oxford Nanopore; single long reads span complete gene regions, presenting alleles and structure in full.

(4) Bioinformatics and interpretation

Using IPD-KIR/IPD-HLA references and a long-read-adapted pipeline, allele-level alignment, structural-variant detection and phase reconstruction yield the GL string, complete centromeric/telomeric haplotypes, copy number, B-content score and KIR–HLA ligand pairing.

Figure 1. Workflow of long-read (PacBio HiFi / Nanopore) KIR genotyping.

3. Features and Advantages

Single-molecule reads across whole genes: Long reads span complete KIR genes, eliminating short-read assembly ambiguity and resolving highly homologous paralogs directly.

Complete phase and haplotypes: Centromeric/telomeric haplotypes are reconstructed directly rather than inferred statistically.

Structural variation accessible: Deletions, duplications and fusions (e.g., KIR3DL1/2v) are presented in full on individual reads.

Highest allele-level resolution: PacBio HiFi provides reference-grade accuracy; the Nanopore long-range-PCR approach is deployable in a routine laboratory, with allele-level concordance of 97.2–99.5%.

Complementary / confirmatory to NGS: Can serve as a confirmation layer for hybrid-capture NGS results, or provide reference-grade typing on its own.

4. Applications

MCB / starting-material confirmation: reference-grade KIR/HLA allele typing and complete haplotype documentation.

High-resolution CAR-NK donor typing: when phase or complete haplotypes are required.

Confirmation of NGS results: long-read confirmation of highly homologous genes and structural variants that short reads cannot resolve.

Novel allele / structural-variant identification: supporting research discovery and database contribution.

IND/BLA characterization: reference-grade immunogenetic evidence for cell-therapy products.

5. Report and Deliverables

The report centers on high-resolution, haplotype-resolved and comparable immunogenetic evidence. Main contents include:

Allele-level genotype: high-resolution genotype in GL string format.

Complete haplotype: centromeric/telomeric (Cen/Tel) and A/B haplotype composition.

Structural variation: presentation of deletions, duplications and fusions (e.g., KIR3DL1/2v).

Copy number: KIR gene copy number based on long reads.

B-content score: calculated from the number of Cen-B and Tel-B motifs (0–4).

KIR–HLA ligand pairing: C1/C2, Bw4 and A3/A11 epitope assignment and mismatch direction.

HLA class I typing: HLA-A/B/C alleles, if co-performed.

Data delivery: full analysis report (PDF), genotype/haplotype list, structural-variant files, and raw long-read data.

Figure 2. Representative results of long-read KIR allele-level genotype, haplotype and structural variation.

6. Service Workflow

*Standard turnaround: approximately 25–35 working days; batch samples and add-on items are subject to the final plan.

7. Sample Requirements

*Detailed requirements follow the latest Sample Submission Guide; long reads are sensitive to DNA integrity, and this service is not suitable for severely degraded samples. Please schedule and confirm the plan before shipping.

8. Technical Specifications

9. References

[1] Downing J, et al. Allele-level KIR genotyping by long-read Oxford Nanopore sequencing. HLA. 2025;106(3):e70400. doi:10.1111/tan.70400

[2] Downing J, D'Orsogna L. High-resolution human KIR genotyping. Immunogenetics. 2022;74(4):369–379. doi:10.1007/s00251-021-01247-0

[3] Norman PJ, et al. Defining KIR and HLA Class I Genotypes at Highest Resolution via High-Throughput Sequencing. Am J Hum Genet. 2016;99(2):375–391. doi:10.1016/j.ajhg.2016.06.023

[4] Cooley S, Weisdorf DJ, Guethlein LA, et al. Donor selection for natural killer cell receptor genes leads to superior survival after unrelated transplantation for acute myelogenous leukemia. Blood. 2010;116(14):2411–2419. doi:10.1182/blood-2010-05-283051

[5] Ruggeri L, Capanni M, Urbani E, et al. Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science. 2002;295(5562):2097–2100. doi:10.1126/science.1068440

[6] Liu E, Marin D, Banerjee P, et al. Use of CAR-Transduced Natural Killer Cells in CD19-Positive Lymphoid Tumors. N Engl J Med. 2020;382(6):545–553. doi:10.1056/NEJMoa1910607

[7] Milius RP, Mack SJ, Hollenbach JA, et al. Genotype List String: a grammar for describing HLA and KIR genotyping results in a text string. Tissue Antigens. 2013;82(2):106–112.

[8] ICH. Q2(R2): Validation of Analytical Procedures. Step 4 version, 2023.