title: Development of a high-resolution NGS-based HLA-typing and analysis pipeline.
abstract: The human leukocyte antigen (HLA) complex contains the most polymorphic genes in the human genome. The classical HLA class I and II genes define the specificity of adaptive immune responses. Genetic variation at the HLA genes is associated with susceptibility to autoimmune and infectious diseases and plays a major role in transplantation medicine and immunology. Currently, the HLA genes are characterized using Sanger- or next-generation sequencing (NGS) of a limited amplicon repertoire or labeled oligonucleotides for allele-specific sequences. High-quality NGS-based methods are in proprietary use and not publicly available. Here, we introduce the first highly automated open-kit/open-source HLA-typing method for NGS. The method employs in-solution targeted capturing of the classical class I (HLA-A, HLA-B, HLA-C) and class II HLA genes (HLA-DRB1, HLA-DQA1, HLA-DQB1, HLA-DPA1, HLA-DPB1). The calling algorithm allows for highly confident allele-calling to three-field resolution (cDNA nucleotide variants). The method was validated on 357 commercially available DNA samples with known HLA alleles obtained by classical typing. Our results showed on average an accurate allele call rate of 0.99 in a fully automated manner, identifying also errors in the reference data. Finally, our method provides the flexibility to add further enrichment target regions.
authors.affiliation: Christian-Albrechts-University of Kiel, Institute of Clinical Molecular Biology, Kiel, Germany m.wittig@mucosa.de.
authors.forename: Michael
authors.initials: M
authors.lastname: Wittig
authors.affiliation: Norwegian PSC Research Center, Department of Transplantation Medicine, Division of Cancer Medicine, Surgery and Transplantation, Oslo University Hospital, Rikshospitalet, Oslo, Norway K.G. Jebsen Inflammation Research Center, Institute of Clinical Medicine, University of Oslo, Oslo, Norway Research Institute of Internal Medicine, Division of Cancer Medicine, Surgery and Transplantation, Oslo University Hospital, Oslo, Norway.
authors.forename: Jarl A
authors.initials: JA
authors.lastname: Anmarkrud
authors.affiliation: Christian-Albrechts-University of Kiel, Department of Computer Science, Kiel, Germany.
authors.forename: Jan C
authors.initials: JC
authors.lastname: Kässens
authors.affiliation: Muthesius Academy of Fine Arts and Design, Kiel, Germany.
authors.forename: Simon
authors.initials: S
authors.lastname: Koch
authors.affiliation: Christian-Albrechts-University of Kiel, Institute of Clinical Molecular Biology, Kiel, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Forster
authors.affiliation: Christian-Albrechts-University of Kiel, Institute of Clinical Molecular Biology, Kiel, Germany.
authors.forename: Eva
authors.initials: E
authors.lastname: Ellinghaus
authors.affiliation: Norwegian PSC Research Center, Department of Transplantation Medicine, Division of Cancer Medicine, Surgery and Transplantation, Oslo University Hospital, Rikshospitalet, Oslo, Norway K.G. Jebsen Inflammation Research Center, Institute of Clinical Medicine, University of Oslo, Oslo, Norway Research Institute of Internal Medicine, Division of Cancer Medicine, Surgery and Transplantation, Oslo University Hospital, Oslo, Norway Section of Gastroenterology, Department of Transplantation Medicine, Division of Cancer Medicine, Surgery and Transplantation, Oslo University Hospital, Rikshospitalet, Oslo, Norway.
authors.forename: Johannes R
authors.initials: JR
authors.lastname: Hov
authors.affiliation: Max-Planck Institute for Molecular Genetics, Berlin, Germany.
authors.forename: Sascha
authors.initials: S
authors.lastname: Sauer
authors.affiliation: Christian-Albrechts-University of Kiel, Department of Computer Science, Kiel, Germany.
authors.forename: Manfred
authors.initials: M
authors.lastname: Schimmler
authors.affiliation: University of Lübeck, Institute of Transfusion Medicine, Lübeck, Germany.
authors.forename: Malte
authors.initials: M
authors.lastname: Ziemann
authors.affiliation: University of Lübeck, Institute of Transfusion Medicine, Lübeck, Germany.
authors.forename: Siegfried
authors.initials: S
authors.lastname: Görg
authors.affiliation: Muthesius Academy of Fine Arts and Design, Kiel, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Jacob
authors.affiliation: Norwegian PSC Research Center, Department of Transplantation Medicine, Division of Cancer Medicine, Surgery and Transplantation, Oslo University Hospital, Rikshospitalet, Oslo, Norway K.G. Jebsen Inflammation Research Center, Institute of Clinical Medicine, University of Oslo, Oslo, Norway Research Institute of Internal Medicine, Division of Cancer Medicine, Surgery and Transplantation, Oslo University Hospital, Oslo, Norway Section of Gastroenterology, Department of Transplantation Medicine, Division of Cancer Medicine, Surgery and Transplantation, Oslo University Hospital, Rikshospitalet, Oslo, Norway t.h.karlsen@medisin.uio.no.
authors.forename: Tom H
authors.initials: TH
authors.lastname: Karlsen
authors.affiliation: Christian-Albrechts-University of Kiel, Institute of Clinical Molecular Biology, Kiel, Germany a.franke@mucosa.de.
authors.forename: Andre
authors.initials: A
authors.lastname: Franke
mesh_terms: D000483:Alleles; D006680:HLA Antigens / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans; D017422:Sequence Analysis, DNA / Q000379:methods*; D012984:Software
chemical_list: D006680:HLA Antigens