P054 : GENOTYPING OF HLA NOVEL AND RARE ALLELES USING NEXT GENERATION SEQUENCING ON THE ION TORRENT PERSONNEL GENOME MACHINE (PGM), USING A NOVEL GROUP-SPECIFIC SANGER SEQUENCE BASED TYPING METHOD AS REFERENCE

Abstract

High resolution genotyping of human leukocyte antigen (HLA) class I and II alleles is important for stem cell transplantation, however it is unclear whether polymorphisms outside of exon 2 and 3 are relevant. With the development of Sanger Sequence Based Typing (SSBT) methods, a high degree of polymorphism at the HLA loci has been identified, making allele identification challenging. Allele ambiguities frequently occur because of the inability to set phase and the lack of complete genomic sequences in reference databases. With the introduction of Next Generation Sequencing (NGS), it is now possible to sequence the HLA genes clonally, completely and simultaneously. Using in-house long range PCR-NGS protocols on the Ion PGM, the aim of this study was to confirm and obtain complete gene sequence for submission to the IMGT/HLA database, on samples identified as novel or rare for HLA Class I alleles by traditional short range (exons 2 and 3) SSBT. In addition, the full length sequences generated by NGS were confirmed using the novel full-length, group-specific sequence based typing (SBT) method, which results in hemizygous sequences, developed in Maastricht. For NGS, long range PCR which amplifies the full gene of HLA-A, -B and -C and exon 2 through to exon 3 of HLA Class II genes was performed. Long range amplicons for each individual are pooled for single library preparation using a modified version of the Ion PGM library preparation and 400 bp chemistry sequencing protocol. All data generated was analysed with software provided by Conexio Genomics and Life Technologies. Analysis of NGS-Ion PGM data identified and confirmed HLA Class I novel and rare alleles reported by short range SSBT. Furthermore, the group-specific sequence based typing results also confirmed data obtained by NGS-Ion PGM. In addition, a number of novel polymorphisms, mainly in intronic sequences, were identified by both sequence based typing methods. NGS-Ion PGM sequencing in addition to group-specific sequence based typing allowed the submission of several full length sequences to the IMGT/HLA database. This study shows the strength of NGS as a tool to complete the IMGT/HLA reference database and the continued relevance of SSBT as a reference methodology. This approach is currently been developed and applied to HLA Class II novel and rare alleles.