title: Ultra-high resolution HLA genotyping and allele discovery by highly multiplexed cDNA amplicon pyrosequencing.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Wisconsin National Primate Research Center, University of Wisconsin-Madison, Madison, WI, USA.
authors.forename: Simon M
authors.initials: SM
authors.lastname: Lank
authors.forename: Brittney A
authors.initials: BA
authors.lastname: Golbach
authors.forename: Hannah M
authors.initials: HM
authors.lastname: Creager
authors.forename: Roger W
authors.initials: RW
authors.lastname: Wiseman
authors.forename: Derin B
authors.initials: DB
authors.lastname: Keskin
authors.forename: Ellis L
authors.initials: EL
authors.lastname: Reinherz
authors.forename: Vladimir
authors.initials: V
authors.lastname: Brusic
authors.forename: David H
authors.initials: DH
authors.lastname: O'Connor
mesh_terms: D000483:Alleles; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D018076:DNA, Complementary; D005805:Genes, MHC Class I; D005802:Genes, MHC Class II; D005838:Genotype; D060005:Genotyping Techniques; D006680:HLA Antigens / Q000032:analysis* / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D006680:HLA Antigens