Amibiguities of HLA DRB1 high resolution typing can be resolved by Sanger sequencing

Abstract

Next Generation Sequencing (NGS) appears to be the future of HLA high resolution as it can deliver allele level typing without ambiguities. However, NGS is still new in its development and may require a high volume of samples to be cost effective for most clinical labs. The existing well-established Sanger sequencing may give us the allele level typing to meet the clinical needs if a proper strategy is implemented. Our lab has used a group-specific amplification/sequencing approach for over a decade. The percentage of the ambiguities in Sequence-Based Typing (SBT) has increased over the years due to continuous discovery of new alleles. To resolve DRB1 SBT ambiguities, we have evaluated an approach of adding an additional sequencing step. HLA DRB1 typing was performed by using a set of group-specific primers (Protrans DRB1 S4). Ambiguities observed in S4 SBT were then resolved by performing an additional amplification and/or sequencing with selected primers (Protrans Domino Stone). Sequencing data were analyzed and allele level typing was assigned by using SBT software (SeqPilot, JSI). Six samples that were initially typed with ambiguities using DRB1 S4 reagent were sequenced with Domino Stones primers. Multiple samples were typed with the same HLA-DRB1 ambiguity. All the ambiguities were resolved with an exception for DRB1*12:01 and 12:10 which are different from each other in exon 1 (Table 1). The final typing assignments can be obtained within 3 days. Our typing results suggest that the new approach of adding an additional sequencing step may resolve most of the DRB1 typing ambiguities and deliver the allele level typing needed by transplant clinicians. Studies in resolving ambiguities of HLA DQB1 with Domino Stone primers are under evaluation.Download : Download full-size image