Abstract
Sequencing-based typing (SBT) provides the highest level of resolution for HLA allelic identification. SBT is capable of discriminating all alleles by nucleotide differences within the amplified region. In case the two alleles are different, the obtained heterozygous sequences frequently match with several different genotypes (genotype ambiguities). These genotypes can be discriminated after separation of the alleles by group-specific sequencing primers (GSSP’s). GSSP’s are selected by the Dynamic Ambiguity Resolving Tool (DART) that is part of the HLA analysis software SBTengine. In the most recent IMGT/HLA database version 3.8 the total number of HLA alleles has succeeded 7,670 allele sequences. This is an increase of 1,136 alleles in one year. With the ever increasing number of HLA alleles, also the percentage of ambiguous results increase. To maintain high levels of typing resolution regular GSSP upgrades are required. We developed a set of second generation GSSP’s for HLA class I and class II that can solve the majority of genotype ambiguities. The percentage of unambiguous typings at the 4-digit level was determined for different populations (European-American, Hispanic, Asian, African-American, Chinese, and Japanese), using the IMGT/HLA database version 3.6.0. Differences in GSSP-usage and resolvability were observed between populations. Using second generation SBT excell erator® GSSP’s for HLA-A resulted in 98% unambiguous results, irrespectively of the population analyzed. For HLA-B, the resolution was 91% (Japanese) to 96% (others). For HLA-C, the resolution ranges from 95% (European-American) to 99% (others). For DRB1, the new GSSP’s resolve 99% of all DRB1 genotype ambiguities for all populations. For DQB1 and DPB1, nearly all ambiguities (>99%) can be resolved. With the second generation of GSSP’s almost all HLA ambiguities can be resolved. High resolution typing levels are obtained for all populations analyzed. Westerink: GenDx: Employee. de Jager: GenDx: Employee. Adema: GenDx: Employee. Bacelar: GenDx: Employee. Hilkes: GenDx: Employee. Rozemuller: GenDx: Employee; Stockholder.
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