OR36 NGS Allele-level typing strategy for nine KIR genes

Abstract

Aim Killer-cell Immunoglobulin-like Receptors (KIRs) are encoded by 17 genes in the Leukocyte Receptor Complex (LRC). These genes are highly polymorphic, on the level of structural variants, and copy-number variation. These characteristics make it challenging to design specific amplifications, and to perform reliable analysis of each KIR allele present in a sample. To enable allele-level resolution KIR genotyping, we developed an NGS-based strategy that involves whole gene amplification of KIR genes. Methods We developed KIR-gene specific amplification primers, with specificity for one or more KIR genes (KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR3DS1, KIR3DL1, KIR3DL2, KIR3DL3 and KIR3DP1). Amplicons were processed in the NGSgo® (GenDx) library preparation procedure (GenDx) and paired-end sequenced on an Illumina MiSeq platform (2 × 151 bp). Data was genotyped with NGSengine® software (GenDx), using KIR database IPD-KIR 2.7.1. Results Multiple samples with known and unknown KIR genotypes were analyzed using this new strategy. KIR amplification, sequencing and subsequent typing was successful for the majority of samples. Many samples could be typed unambiguously, despite the fact that phasing was not fully accomplished due to the length of the genes (up to 17 kb). Remaining challenges in analysis are samples with KIR copy number variation and high degree of homology, which can hamper the data analysis. Conclusions KIR genotyping at allelic level is feasible using the short read sequencing technology of Illumina. This new typing strategy may be an attractive alternative to existing KIR genotype assays that only determine KIR gene content at a limited resolution, or complex sequencing methods aiming to sequence the entire LCR. L.A. van de Pasch: 5. Employee; Company/Organization; GenDx. K. van Ham: 5. Employee; Company/Organization; GenDx. S. Vendelbosch: 5. Employee; Company/Organization; GenDx. M.T. Penning: 5. Employee; Company/Organization; GenDx. E.H. Rozemuller: 5. Employee; Company/Organization; GenDx. 6. Stock Shareholder; Company/Organization; GenDx. 7. Other (Identify); Company/Organization; Founder of GenDx.