title: Combining one-step Sanger sequencing with phasing probe hybridization for HLA class I typing yields rapid, G-group resolution predicting 99% of unique full length protein sequences.
abstract: Sanger-based DNA sequencing of exons 2+3 of HLA class I alleles from a heterozygote frequently results in two or more alternative genotypes. This study was undertaken to reduce the time and effort required to produce a single high resolution HLA genotype.
  
  
  
  Samples were typed in parallel by Sanger sequencing and oligonucleotide probe hybridization. This workflow, together with optimization of analysis software, was tested and refined during the typing of over 42,000 volunteers for an unrelated hematopoietic progenitor cell donor registry. Next generation DNA sequencing (NGS) was applied to over 1000 of these samples to identify the alleles present within the G group designations.
  
  
  
  Single genotypes at G level resolution were obtained for over 95% of the loci without additional assays. The vast majority of alleles identified (>99%) were the primary allele giving the G groups their name. Only 0.7% of the alleles identified encoded protein variants that were not detected by a focus on the antigen recognition domain (ARD)-encoding exons.
  
  
  
  Our combined method routinely provides biologically relevant typing resolution at the level of the ARD. It can be applied to both single samples or to large volume typing supporting either bone marrow or solid organ transplantation using technologies currently available in many HLA laboratories.
authors.affiliation: Departments of Oncology and Pediatrics, Georgetown University, Washington, District of Columbia.
authors.forename: Bin
authors.initials: B
authors.lastname: Tu
authors.affiliation: Departments of Oncology and Pediatrics, Georgetown University, Washington, District of Columbia.
authors.forename: Carly
authors.initials: C
authors.lastname: Masaberg
authors.affiliation: Departments of Oncology and Pediatrics, Georgetown University, Washington, District of Columbia.
authors.forename: Lihua
authors.initials: L
authors.lastname: Hou
authors.affiliation: Data Blueprint, Richmond, Virginia.
authors.forename: Daniel
authors.initials: D
authors.lastname: Behm
authors.affiliation: One Lambda Inc. (Thermo Fisher Scientific), Canoga Park, California.
authors.forename: Peter
authors.initials: P
authors.lastname: Brescia
authors.affiliation: Departments of Oncology and Pediatrics, Georgetown University, Washington, District of Columbia.
authors.forename: Nuri
authors.initials: N
authors.lastname: Cha
authors.affiliation: Departments of Oncology and Pediatrics, Georgetown University, Washington, District of Columbia.
authors.forename: Kanthi
authors.initials: K
authors.lastname: Kariyawasam
authors.affiliation: One Lambda Inc. (Thermo Fisher Scientific), Canoga Park, California.
authors.forename: Jar How
authors.initials: JH
authors.lastname: Lee
authors.affiliation: One Lambda Inc. (Thermo Fisher Scientific), Canoga Park, California.
authors.forename: Thoa
authors.initials: T
authors.lastname: Nong
authors.affiliation: Data Blueprint, Richmond, Virginia.
authors.forename: John
authors.initials: J
authors.lastname: Sells
authors.affiliation: One Lambda Inc. (Thermo Fisher Scientific), Canoga Park, California.
authors.forename: Paul
authors.initials: P
authors.lastname: Tausch
authors.affiliation: Departments of Oncology and Pediatrics, Georgetown University, Washington, District of Columbia.
authors.forename: Ruyan
authors.initials: R
authors.lastname: Yang
authors.affiliation: Departments of Oncology and Pediatrics, Georgetown University, Washington, District of Columbia.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Ng
authors.affiliation: Departments of Oncology and Pediatrics, Georgetown University, Washington, District of Columbia.
authors.forename: Carolyn Katovich
authors.initials: CK
authors.lastname: Hurley
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D005091:Exons; D005838:Genotype; D018380:Hematopoietic Stem Cell Transplantation; D059014:High-Throughput Nucleotide Sequencing / Q000295:instrumentation / Q000379:methods*; D015395:Histocompatibility Antigens Class I / Q000145:classification / Q000235:genetics* / Q000276:immunology; D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes / Q000737:chemistry; D012042:Registries; D061349:Unrelated Donors
chemical_list: D015395:Histocompatibility Antigens Class I; D015345:Oligonucleotide Probes
keywords: 
  DNA probes; 
  DNA sequencing; genetic polymorphism; histocompatibility testing; human leukocyte antigens