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

Simon M. Lank, Brittney A. Golbach, Hannah M. Creager, Roger W. Wiseman, Derin B. Keskin, Ellis L. Reinherz, Vladimir Brusic, David H. O'Connor

Research output: Contribution to journalArticle

23 Citations (Scopus)

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.

Original languageEnglish
Article number378
JournalBMC Genomics
Volume13
Issue number1
DOIs
Publication statusPublished - Aug 6 2012
Externally publishedYes

Fingerprint

Dichlororibofuranosylbenzimidazole
Complementary DNA
Alleles
Galaxies
Genotype
MHC Class II Genes
Workflow
Open Reading Frames
Exons
Databases
Research
Population

Keywords

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

ASJC Scopus subject areas

  • Biotechnology
  • Genetics

Cite this

Lank, S. M., Golbach, B. A., Creager, H. M., Wiseman, R. W., Keskin, D. B., Reinherz, E. L., ... O'Connor, D. H. (2012). Ultra-high resolution HLA genotyping and allele discovery by highly multiplexed cDNA amplicon pyrosequencing. BMC Genomics, 13(1), [378]. https://doi.org/10.1186/1471-2164-13-378

Ultra-high resolution HLA genotyping and allele discovery by highly multiplexed cDNA amplicon pyrosequencing. / Lank, Simon M.; Golbach, Brittney A.; Creager, Hannah M.; Wiseman, Roger W.; Keskin, Derin B.; Reinherz, Ellis L.; Brusic, Vladimir; O'Connor, David H.

In: BMC Genomics, Vol. 13, No. 1, 378, 06.08.2012.

Research output: Contribution to journalArticle

Lank, SM, Golbach, BA, Creager, HM, Wiseman, RW, Keskin, DB, Reinherz, EL, Brusic, V & O'Connor, DH 2012, 'Ultra-high resolution HLA genotyping and allele discovery by highly multiplexed cDNA amplicon pyrosequencing', BMC Genomics, vol. 13, no. 1, 378. https://doi.org/10.1186/1471-2164-13-378
Lank, Simon M. ; Golbach, Brittney A. ; Creager, Hannah M. ; Wiseman, Roger W. ; Keskin, Derin B. ; Reinherz, Ellis L. ; Brusic, Vladimir ; O'Connor, David H. / Ultra-high resolution HLA genotyping and allele discovery by highly multiplexed cDNA amplicon pyrosequencing. In: BMC Genomics. 2012 ; Vol. 13, No. 1.
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