title: Rapid, scalable and highly automated HLA genotyping using next-generation sequencing: a transition from research to diagnostics.
abstract: BACKGROUND
  Human leukocyte antigen matching at allelic resolution is proven clinically significant in hematopoietic stem cell transplantation, lowering the risk of graft-versus-host disease and mortality. However, due to the ever growing HLA allele database, tissue typing laboratories face substantial challenges. In light of the complexity and the high degree of allelic diversity, it has become increasingly difficult to define the classical transplantation antigens at high-resolution by using well-tried methods. Thus, next-generation sequencing is entering into diagnostic laboratories at the perfect time and serving as a promising tool to overcome intrinsic HLA typing problems. Therefore, we have developed and validated a scalable automated HLA class I and class II typing approach suitable for diagnostic use.
  
  
  RESULTS
  A validation panel of 173 clinical and proficiency testing samples was analysed, demonstrating 100% concordance to the reference method. From a total of 1,273 loci we were able to generate 1,241 (97.3%) initial successful typings. The mean ambiguity reduction for the analysed loci was 93.5%. Allele assignment including intronic sequences showed an improved resolution (99.2%) of non-expressed HLA alleles.
  
  
  CONCLUSIONS
  We provide a powerful HLA typing protocol offering a short turnaround time of only two days, a fully integrated workflow and most importantly a high degree of typing reliability. The presented automated assay is flexible and can be scaled by specific primer compilations and the use of different 454 sequencing systems. The workflow was successfully validated according to the policies of the European Federation for Immunogenetics. Next-generation sequencing seems to become one of the new methods in the field of Histocompatibility.
authors.affiliation: Department of Immunogenetics, Red Cross Transfusion Service for Upper Austria, Krankenhausstrasse 7, Linz 4017, Austria. martin.danzer@o.roteskreuz.at
authors.forename: Martin
authors.initials: M
authors.lastname: Danzer
authors.forename: Norbert
authors.initials: N
authors.lastname: Niklas
authors.forename: Stephanie
authors.initials: S
authors.lastname: Stabentheiner
authors.forename: Katja
authors.initials: K
authors.lastname: Hofer
authors.forename: Johannes
authors.initials: J
authors.lastname: Pröll
authors.forename: Christina
authors.initials: C
authors.lastname: Stückler
authors.forename: Edeltraud
authors.initials: E
authors.lastname: Raml
authors.forename: Helene
authors.initials: H
authors.lastname: Polin
authors.forename: Christian
authors.initials: C
authors.lastname: Gabriel
mesh_terms: D057205:Automation, Laboratory / Q000379:methods; D006680:HLA Antigens / Q000032:analysis*; D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans; D058885:Multilocus Sequence Typing / Q000379:methods; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D015203:Reproducibility of Results
chemical_list: D006680:HLA Antigens
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