Open Access Highly Accessed Open Badges Methodology article

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

Simon M Lank1, Brittney A Golbach1, Hannah M Creager1, Roger W Wiseman1, Derin B Keskin23, Ellis L Reinherz23, Vladimir Brusic23 and David H O’Connor14*

Author affiliations

1 Wisconsin National Primate Research Center, University of Wisconsin-Madison, Madison, WI, USA

2 Cancer Vaccine Center, DanaFarber Cancer Institute, Boston, MA, USA

3 Department of Medicine, Harvard Medical School, Boston, MA, USA

4 Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, WI, USA

For all author emails, please log on.

Citation and License

BMC Genomics 2012, 13:378  doi:10.1186/1471-2164-13-378

Published: 6 August 2012



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 a lack of locus coverage and difficulty in exon-phase matching. Achieving high-resolution typing is also becoming more challenging with traditional methods as the database of known HLA alleles increases.


We designed a cDNA amplicon-based pyrosequencing method to capture 94% of the HLA class I open-reading-frame with only two amplicons per sample, and an analogous method for class II HLA genes, with a primary focus on sequencing the DRB loci. We present a novel Galaxy server-based analysis workflow for determining genotype. During assay validation, we performed two GS Junior sequencing runs to determine the accuracy of the HLA class I amplicons and DRB amplicon at different levels of multiplexing. When 116 amplicons were multiplexed, we unambiguously resolved 99%of class I alleles to four- or six-digit resolution, as well as 100% unambiguous DRB calls. The second experiment, with 271 multiplexed amplicons, missed some alleles, but generated high-resolution, concordant typing for 93% of class I alleles, and 96% for DRB1 alleles. In a third, preliminary experiment we attempted to sequence novel amplicons for other class II loci with mixed success.


The presented assay is higher-throughput and higher-resolution than existing HLA genotyping methods, and suitable for allele discovery or large cohort sampling. The validated class I and DRB primers successfully generated unambiguously high-resolution genotypes, while further work is needed to validate additional class II genotyping amplicons.

HLA; Genotyping; Roche/454; Pyrosequencing; Galaxy; Tissue typing; Cellular immunity; Multiplexing