OR06: GROUP SPECIFIC, UNAMBIGUOUS FULL-LENGTH GENE HLA CLASS I TYPING BY SANGER SEQUENCING: A ROBUST SSBT STRATEGY FOR TYPING AND A GOLDEN REFERENCE FOR NEXT GENERATION SEQUENCING APPROACHES

Abstract

Classically, sequence base typing (SBT) of HLA is performed using sequences from exons 2, 3 and 4 for HLA class I and exons 2 and 3 for HLA class II. This partial analysis of HLA genes usually leads to allele and genotype ambiguities, which causes complications in the assignment of alleles. Consequently, the current IMGT database lacks complete sequences for more than 90% of all alleles. We developed and validated a group specific Sanger sequencing approach that results in an unambiguous typing result for HLA class I alleles. This approach provides full length gene sequences and enables not only an unambiguous typing of alleles, but also allows for filling sequence gaps in the unknown allele sequences of the IMGT/HLA database. The method is based on group specific, full-length amplification of HLA class I alleles using any low resolution results. The alleles were separated into 8 different groups for HLA-A, 15 for HLA-B and 13 for HLA-C. Depending on the location of the group-specific primers, amplicons varied in length between 2.9 and 3.2 kb for HLA-A, 2.8 and 3.8 kb for HLA-B and 2.8 and 3.3 kb for HLA-C. The amplicon is sequenced using generic sequencing primers, suitable for all amplified groups. This approach is routinely applicable for high resolution typing of clinical samples and the identification of allele specific full length gene sequences, with some additional sequence primers. Currently we confirmed the complete allele sequences of HLA-A∗02:01:01:01, A∗03:01:01:01, A31:01:02, B∗08:01:01, B∗15:01:01:01, B∗40:01:01, B∗51:01:01, C∗04:01:01:01, C∗05:01:01:01, C∗07:01:01:01 and C∗12:02:02, and identified full length sequences of partially known (e.g. HLA-A∗74:11) and novel alleles (e.g. HLA-A∗23:38 N, B∗35:241 or C∗02:69). Moreover, this full-length group specific approach can serve as THE reference for next generation sequencing approaches where new polymorphism in introns and all exons, previously unknown in the IMGT/HLA database, are identified.