P108 Phased whole-gene characterization of novel KIR3DL1, KIR3DL2, and KIR3DL3 alleles using a dual redundant sequencing strategy

Aim The human killer-cell immunoglobulin-like receptors (KIR) forms a complex receptor family expressed on the surface of natural killer (NK) cells. KIR genotypes have been demonstrated to affect disease predisposition and transplantation outcome. To facilitate the selection of donors based on KIR genotypes, we added KIR to the genotyping profile for newly registered potential stem cell donors in 2014. Up to now we have KIR genotyped more than 2 million samples based on our next-generation sequencing approach. This resulted in the discovery of thousands of novel alleles. For the submission of novel alleles to the IPD-KIR Database, full-length phased sequence characterization is desirable. Therefore, we initiated a project for high quality full-length sequence characterization of novel KIR3DL1 , KIR3DL2 , and KIR3DL3 alleles. Methods We established three long-range PCRs targeting the whole genes including both UTRs resulting in PCR products of 13.5 kb ( KIR3DL1 ), 17.5 kb ( KIR3DL2 ), and 16 kb ( KIR3DL3 ). Redundant sequencing strategies were applied to sequence two independent PCR products per target: Shotgun sequencing on Illumina MiSeq instruments and Single Molecule Real Time (SMRT) sequencing on Pacific Biosciences Sequel Systems. Using the Dual Redundant Reference Sequencing software (DR2S, DKMS Life Science Lab), phase-defined consensus sequences were generated from the SMRT data and confirmed with the short, high-fidelity reads. Applying this approach we analyzed a set of 364 samples targeting 112 distinct novel KIR alleles (41 KIR3DL1 , 37 KIR3DL2 , and 34 KIR3DL3 ). Results The generated sequencing data revealed high specificity of the assays for KIR3DL1 , KIR3DL2 and KIR3DL3 . The approach proved capable of generating fully phased sequences spanning the entire 13 to 17 kb genes. By applying two complementary technologies, rare sequencing errors could be corrected. This enabled high-quality phase-defined characterization of novel KIR alleles without the requirement for time consuming cloning steps. Conclusions We established a method for highest quality phased whole-gene KIR sequencing and applied it to characterize more than 100 novel KIR3DL1 , KIR3DL2 , and KIR3DL3 alleles. By submitting these sequences to IPD-KIR we will complement the database for frequent but so far undescribed KIR alleles.