P185 Next generation sequencing of HLA class II in an argentinian registry population reveals the complexity of these loci

Aim This study used next generation sequencing (NGS) in 1472 healthy volunteers from an Argentinian registry to characterize class II gene diversity. Complications of using NGS for these regions arise from large introns, different length alleles, regions with few polymorphisms, and incomplete sequences in the IPD-IMGT/HLA database. Methods Amplicons were generated to include exons 2–6 of HLA-DQB1, and exons 2–3 of -DRB1 and -DPB1 using DNA extracted from blood. Samples were combined and sequenced on an Illumina MiSeq with an in-house library construction protocol. Conexio Assign was used to identify alleles based on the IPD-IMGT/HLA database from October 2016. Full length gene sequencing of select samples was performed with two overlapping amplicons for HLA-DPB1 and -DRB1. Results The majority of the alleles identified at each locus were common with ⩽1% of alleles per gene designated as well-documented or with frequency not defined. With only exons 2–3 sequenced, 6% of HLA-DRB1 assignments and 11% of -DPB1 were ambiguous. When additional exons were sequenced, most showed the common allele; however, DPB1∗104:01 was seen in 16% of samples typed as DPB1∗03:FNVX. For HLA-DRB1, preferential amplification due to differences in allele size and presence of pseudogenes led to certain alleles with low read depth and poor sequence quality, in particular DRB1∗04. Analysis of HLA-DQB1 introns proved difficult due to insertions and deletions that poorly aligned to the reference. Intron 2 of HLA-DPB1 is large and has few variations between certain combinations of alleles that were not phased in 14% of samples. These were not resolved with full length sequencing. Conclusions Although conditions still need to be optimized, NGS of HLA class II is a powerful tool in determining genetic diversity. HLA-DRB1 ambiguities would be reduced by 82% with the initial sequencing of exons 2–4; however, longer amplicons reduce read depth. Addition of primers to maximize amplification of low amplifying alleles may be necessary to increase read depth. Alternate methodology such as sequencing of single DNA molecules is necessary to solve the ambiguities for alleles that are not phased. These proposed changes will lead to more successful sequencing of HLA class II. Even so, these complications make NGS challenging when applied to high volume typing.