P205 Completing a rare HLA-DRB1*13 genomic allele sequence based on long range amplicons and next generation sequencing

Aim Some years ago we found a new HLA-DRB1∗13 allele in a family in Germany with roots in Turkey and Armenia. It was found in a male potential stem cell donor, in his own and in his extended family. Sanger sequencing of exon 2 of this allele, later called DRB1∗13:54, revealed 3 nucleotide variations at position 157 (T → A), 158 (C → T) and 166 (C → A). Methods To complete the genomic sequence and to clarify the degree of recombination within this allele, we developed a workflow based on long range PCR (LR-PCR) and next generation sequencing (NGS). Therefore we designed different HLA locus and/or allele specific LR-PCRs and sequenced the generated amplicons on a MiSeq platform (Illumina). The subsequent NGS data evaluation was performed with two different HLA software tools (Omixon Twin, Omixon and NGSengine, GenDx). The phased sequence alignment according to the individual single nucleotide variants (SNVs) pattern present ended up with allele-specific contigs. The final alignment of these contigs was done with AliView (Muscle) and BioEdit (ClustalW) software along with published IMGT/HLA database sequences. Results The full-length sequence analysis of the described allele unraveled a quite high similarity to the DRB1∗13:24 allele. The evolutionary history of the DRB1∗13:54 could be best explained by a recombination of DRB1∗13:04 (acceptor) with DRB1∗07 (∗07:01-∗07:04, ∗07:07) or DRB1∗12 (∗12:01, ∗12:03, ∗12:05-∗12:08) as donor. Conclusions With our approach, applying LR-PCR and NGS including phased sequence analysis, we were able to determine the complete gene sequence of HLA-DRB1∗13:54 (from 5’-UTR to 3’-UTR). The most likely evolutionary recombination between the DRB1∗13 allele and the DRB1∗07 or DRB1∗12 alleles seems to be restricted to exon 2 only.