High resolution NGS-based HLA-typing using in-solution targeted enrichment

Michael Wittig1, Jarl Andreas Anmarkrud2,3,4, Jan Christian Kässens5, Simon Koch6, Michael Forster1, Eva Ellinghaus1, Johannes E.R. Hov2,4,7, Sascha Sauer8, Manfred Schimmler5, Malte Ziemann9, Siegfried Görg9, Frank Jacob6, Tom Hemming Karlsen2,4,7, Andre Franke1 1Christian-Albrechts-University Kiel, Institute of Clinical Molecular Biology, Kiel, Germany;  2Norwegian PSC Research Center, Department of Transplantation Medicine, Divison of Cancer Medicine, Surgery and Transplantation, Oslo University Hospital, Rikshospitalet, Oslo, Norway;  3K.G. Jebsen Inflammation Research Center, Institute of Clinical Medicine, University of Oslo, Oslo, Norway;  4Research Institute of Internal Medicine, Division of Cancer Medicine, Surgery and Transplantation, Oslo University Hospital, Oslo, Norway;  5Christian-Albrechts-University of Kiel, Department of Computer Science, Kiel, Germany;  6Muthesius Academy of Fine Arts and Design, Kiel, Germany; 7Section of Gastroenterology, Department of Transplantation Medicine, Division of Cancer Medicine, Surgery and Transplantation, Oslo University Hospital, Rikshospitalet, Oslo, Norway;  8Max-Planck Institute for Molecular Genetics, Berlin;  9University of Lübeck, Institute of Transfusion Medicine, Lübeck, Germany

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 organ transplantation 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. We developed a highly automated 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, HLADQA1, HLA-DQB1, HLA-DPA1, HLA-DPB1) followed by an amplification of the captured DNAs. The method was tested 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, the method provides the flexibility to add further enrichment target regions.