Development of Super high resolution Single moleculeSequence Based Typing (SS-SBT) method in HLA-DRB1 gene by next generation sequencing

Abstract

Current HLA-DNA typing methods, PCR-SBT and PCR-Luminex methods, are used for routine testing. However, these methods yield ambiguous typing results because of lack of oligonucleotide probes and phase ambiguity for HLA allele determination. In this paper we described the development and first application of 8-digit level super high resolution single molecule-sequence based typing (SS-SBT) method for HLA-DRB1 loci using next generation sequencer, Roche GS Junior Bench Top System aimed at elimination of phase ambiguity. Seven PCR group-specific primer pairs for HLA-DRB1 were designed to amplify the genomic region from exon 2 to exon 6, and the PCR condition was set to amplify all of the HLA-DRB1 alleles simultaneously in a single tube. By this SS-SBT typing of the HLA-DRB1 loci using 19 samples that were observed with phase ambiguities for HLA-DRB1 when defined by conventional HLA-DNA typing methods, all of them except for two alleles were typed to single HLA alleles at the 8-digit level by next generation sequencing. Therefore, the SS-SBT method is strongly suggested to be a superior HLA-DNA typing method to efficiently detect new HLA alleles and null alleles along with effective 8-digit level DNA typing for HLA-DRB1 without ambiguity.