P223 Adding class II to a one step Sanger sequencing with phasing probe hybridization workflow_a rapid and complete HLA typing at G-group resolution

Aim In order to reduce the time and effort required to produce a single high resolution HLA class I genotype, a workflow combining one-step Sanger sequencing with probe hybridization has been successfully used in HLA-A, -B, -C typing for a hematopoietic stem cell donor registry (Tu et al. HLA 89:90, 2017). This workflow has now been applied to type HLA-DRB1, -DQB1 and -DPB1. Methods DNA was prepared from buccal swabs from 184 registry donors. Sanger sequencing used Thermo Fisher Scientific SeCore® HLA-DRB1, -DQB1, and -DPB1 kits. Simultaneous hybridization used One Lambda LABType® probes. Assignments were merged with in-house software. Interpretation used IPD-IMGT/HLA database 3.19. Results Use of both DNA sequencing and SSO (without additional phasing probes) hybridization resulted in assignment of a single G-level genotype for the majority of samples compared to sequencing alone (Table). Ambiguous genotypes observed for DRB1 and DQB1 will be addressed by additional phasing probes to raise the frequency of single genotype resolution for these loci closer to 100%. Conclusions This workflow complements that described by us for HLA class I alleles and provides a comprehensive strategy for complete HLA typing. The SBT+SSO combination workflow with genotype assignments at the level of the antigen recognition domain is a powerful typing strategy, merging strengths of both methods and improving accuracy and reducing turnaround time and typing cost. While applied to registry typing, this workflow also can be applied to a small number of samples. Download high-res image (155KB) Download full-size image