title: Application of High-Throughput Next-Generation Sequencing for HLA Typing on Buccal Extracted DNA: Results from over 10,000 Donor Recruitment Samples.
abstract: BACKGROUND
  Unambiguous HLA typing is important in hematopoietic stem cell transplantation (HSCT), HLA disease association studies, and solid organ transplantation. However, current molecular typing methods only interrogate the antigen recognition site (ARS) of HLA genes, resulting in many cis-trans ambiguities that require additional typing methods to resolve. Here we report high-resolution HLA typing of 10,063 National Marrow Donor Program (NMDP) registry donors using long-range PCR by next generation sequencing (NGS) approach on buccal swab DNA.
  
  
  METHODS
  Multiplex long-range PCR primers amplified the full-length of HLA class I genes (A, B, C) from promotor to 3' UTR. Class II genes (DRB1, DQB1) were amplified from exon 2 through part of exon 4. PCR amplicons were pooled and sheared using Covaris fragmentation. Library preparation was performed using the Illumina TruSeq Nano kit on the Beckman FX automated platform. Each sample was tagged with a unique barcode, followed by 2×250 bp paired-end sequencing on the Illumina MiSeq. HLA typing was assigned using Omixon Twin software that combines two independent computational algorithms to ensure high confidence in allele calling. Consensus sequence and typing results were reported in Histoimmunogenetics Markup Language (HML) format. All homozygous alleles were confirmed by Luminex SSO typing and exon novelties were confirmed by Sanger sequencing.
  
  
  RESULTS
  Using this automated workflow, over 10,063 NMDP registry donors were successfully typed under high-resolution by NGS. Despite known challenges of nucleic acid degradation and low DNA concentration commonly associated with buccal-based specimens, 97.8% of samples were successfully amplified using long-range PCR. Among these, 98.2% were successfully reported by NGS, with an accuracy rate of 99.84% in an independent blind Quality Control audit performed by the NDMP. In this study, NGS-HLA typing identified 23 null alleles (0.023%), 92 rare alleles (0.091%) and 42 exon novelties (0.042%).
  
  
  CONCLUSIONS
  Long-range, unambiguous HLA genotyping is achievable on clinical buccal swab-extracted DNA. Importantly, full-length gene sequencing and the ability to curate full sequence data will permit future interrogation of the impact of introns, expanded exons, and other gene regulatory sequences on clinical outcomes in transplantation.
authors: UCLA Immunogenetics Center, Department of Pathology & Laboratory Medicine, Los Angeles, CA, United States of America.
authors: Yuxin
authors: Y
authors: Yin
authors: University of British Columbia Clinician Investigator Program, Vancouver, BC, Canada.
authors: James H
authors: JH
authors: Lan
authors: UCLA Immunogenetics Center, Department of Pathology & Laboratory Medicine, Los Angeles, CA, United States of America.
authors: David
authors: D
authors: Nguyen
authors: UCLA Immunogenetics Center, Department of Pathology & Laboratory Medicine, Los Angeles, CA, United States of America.
authors: Nicole
authors: N
authors: Valenzuela
authors: UCLA Immunogenetics Center, Department of Pathology & Laboratory Medicine, Los Angeles, CA, United States of America.
authors: Ping
authors: P
authors: Takemura
authors: National Marrow Donor Program, Minneapolis, MN, United States of America.
authors: Yung-Tsi
authors: YT
authors: Bolon
authors: National Marrow Donor Program, Minneapolis, MN, United States of America.
authors: Brianna
authors: B
authors: Springer
authors: One Lambda, Thermo Fisher Scientific, Canoga Park, CA, United States of America.
authors: Katsuyuki
authors: K
authors: Saito
authors: UCLA Immunogenetics Center, Department of Pathology & Laboratory Medicine, Los Angeles, CA, United States of America.
authors: Ying
authors: Y
authors: Zheng
authors: Omixon LTD, Budapest, Hungary.
authors: Tim
authors: T
authors: Hague
authors: Omixon LTD, Budapest, Hungary.
authors: Agnes
authors: A
authors: Pasztor
authors: Omixon LTD, Budapest, Hungary.
authors: Gyorgy
authors: G
authors: Horvath
authors: Omixon LTD, Budapest, Hungary.
authors: Krisztina
authors: K
authors: Rigo
authors: UCLA Immunogenetics Center, Department of Pathology & Laboratory Medicine, Los Angeles, CA, United States of America.
authors: Elaine F
authors: EF
authors: Reed
authors: UCLA Immunogenetics Center, Department of Pathology & Laboratory Medicine, Los Angeles, CA, United States of America.
authors: Qiuheng
authors: Q
authors: Zhang
mesh_terms: D000483:Alleles; D005091:Exons; D005787:Gene Frequency; D005838:Genotype; D006680:HLA Antigens / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006650:Histocompatibility Testing / Q000379:methods; D006801:Humans; D009061:Mouth Mucosa / Q000378:metabolism*; D060885:Multiplex Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014019:Tissue Donors; D057188:Workflow
chemical_list: D006680:HLA Antigens