P087 A next generation sequencing based genotyping strategy for HLA-E

Aim In 2013, we implemented a short amplicon based Next Generation Sequencing (NGS) high-throughput workflow for HLA donor registry typing. Data analysis is performed by the in-house typing software neXtype. Currently, over 30,000 samples per week can be typed for 6 HLA loci, ABO, RhD, CCR5, and KIR with this cost-efficient workflow in our lab. As HLA-E has been reported to impact the outcome of stem cell transplantations in certain settings, we evaluated the feasibility of low-cost high-throughput HLA-E typing. On a clinically relevant antigen recognition domain (ARD) level, there are currently 9 groups of HLA-E alleles known (Fig. 1). Methods HLA-E is amplified by PCR with two primers spanning exons 2 and 3. This amplification product is subject to sequencing on Illumina MiSeq or HiSeq 2500 instruments without fragmentation. Thereby, information about the phasing between exon 2 and exon 3 is preserved, which is an important advantage of this method since it allows genotyping at ARD-level resolution. As shown in Fig. 1, alleles HLA-E∗01:01:02, 01:03:03, 01:04, 01:05, 01:07 and 01:08N can be resolved to the allele level. The remaining alleles fall into three distinguishable G-groups, namely HLA-E∗01:01:01G, 01:03:01G and 01:03:02G. Results The primer set was tested using 384 samples. All successfully sequenced samples corresponded to the G-groups HLA-E∗01:01:01G, 01:03:01G and 01:03:02G. Validation of the workflow against pre-typed samples is projected. Conclusions Sequencing HLA-E on allelic level has been show feasible in a cost efficient high throughput workflow. As the impact of HLA-E on the transplantation outcome becomes more settled, it could be considered for inclusion into the recruitment typing profile. Download high-res image (244KB) Download full-size image