Ultra-high resolution HLA genotyping and allele discovery by highly multiplexed cDNA amplicon pyrosequencing

摘要: High-resolution HLA genotyping is a critical diagnostic and research assay. Current methods rarely achieve unambiguous high-resolution typing without making population-specific frequency inferences due to lack of locus coverage difficulty in exon-phase matching. Achieving also becoming more challenging with traditional as the database known alleles increases. We designed cDNA amplicon-based pyrosequencing method capture 94% class I open-reading-frame only two amplicons per sample, an analogous for II genes, primary focus on sequencing DRB loci. present novel Galaxy server-based analysis workflow determining genotype. During assay validation, we performed GS Junior runs determine accuracy amplicon at different levels multiplexing. When 116 were multiplexed, unambiguously resolved 99%of four- or six-digit resolution, well 100% calls. The second experiment, 271 multiplexed amplicons, missed some alleles, but generated high-resolution, concordant 93% 96% DRB1 alleles. In third, preliminary experiment attempted sequence other loci mixed success. presented higher-throughput higher-resolution than existing methods, suitable allele discovery large cohort sampling. validated primers successfully genotypes, while further work needed validate additional amplicons.