OR02: DEVELOPMENT OF ADVANCED NGS BASED HLA DNA TYPING METHOD: SS-SBT.

Aim Super high resolution-single molecule-sequence-based typing (SS-SBT) is an HLA DNA typing method to the field 4 level of allelic resolution (8-digit typing) by combination of long ranged PCR amplification and next generation sequencing (NGS) technologies. We previously reported the development and application of the SS-SBT method such as long ranged PCR system from promoter-enhancer to 3’UTR for the 11 classical HLA loci, A, B, C, DRB1, DRB3/4/5, DQA1, DQB1, DPA1 and DPB1, setting of NGS parameters, and automated NGS data processing system, Sequence Alignment Based Assigning Software (SeaBass). Here we report the development of the advanced SS-SBT method. Methods (1) Since allelic imbalance was observed in some particular alleles, the PCR primer sequences were modified for some HLA loci. (2) We newly designed middle ranged PCR primer sets for amplification from intron 1 to exon 4 in DRB1, DQB1 and DPB1 (3.8 kb to 5.3 kb). (3) We improved the program (SeaBass) to avoid allele mis-assignment. Results (1) By application of the SS-SBT using over 300 DNA samples that were observed with phase ambiguities when defined by conventional HLA DNA typing methods (SBT, PCR-SSO etc.), all of them, except for DQA1, DPA1 and DPB1, were typed to single HLA alleles to the field 4 level with minimum allelic imbalance. (2) The new PCR system including primers previously designed A, B and C (4.6 kb to 5.5 kb) is specialized to typing for polymorphic exons (the field 3 level or 6-digit typing), contributing to a convenient replacement model from conventional typing methods to NGS based typing method. (3) In comparison of three different automated NGS data processing systems such as Omixon Target 1.6.0 and NGSengine 1.2, SeaBass could call all of HLA alleles assigned by conventional methods with 100% accuracy. Conclusion The advanced SS-SBT method is a powerful tool that provides precise allele data for efficient detection of new and null alleles without phase ambiguity. [ABSTRACT FROM AUTHOR]