Tissue Antigens ISSN 0001-2815 Super high resolution for single molecule-sequence-based typing of classical HLA loci at the 8-digit level using next generation sequencers T. Shiina 1,∗ , S. Suzuki 1,∗ , Y. Ozaki 1,∗ , H. Taira 2 , E. Kikkawa 1 , A. Shigenari 1 , A. Oka 1 , T. Umemura 3 , S. Joshita 3 , O. Takahashi 2 , Y. Hayashi 4 , M. Paumen 4 , Y. Katsuyama 5 , S. Mitsunaga 1 , M. Ota 6 , J. K. Kulski 7 & H. Inoko 1 1 Division of Basic Medical Science and Molecular Medicine, Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Kanagawa, Japan 2 Technical Support, Life Technologies Japan Ltd, Minato-ku, Tokyo, Japan 3 Division of Gastroenterology and Hepatology, Department of Medicine, Shinhsu University School of Medicine, Matsumoto, Nagano, Japan 4 Scientific Affairs, Life Technologies Japan Ltd, Minato-ku, Tokyo, Japan 5 Department of Pharmacy, Shinshu University Hospital, Matsumoto, Nagano, Japan 6 Department of Legal Medicine, Shinshu University School of Medicine, Matsumoto, Nagano, Japan 7 Centre for Forensic Science, The University of Western Australia, Nedlands, Western Australia, Australia Key words DNA typing; human leukocyte antigen; next generation sequencing; polymerase chain reaction; super high-resolution single-molecule sequence-based typing Correspondence Hidetoshi Inoko, PhD, Department of Molecular Life Science Division of Basic Medical Science and Molecular Medicine Tokai University School of Medicine Isehara Kanagawa 259-1143 Japan Tel: +81 463 93 1121 Fax: +81 463 94 8884 e-mail: hinoko@is.icc.u-tokai.ac.jp Received 22 May 2012; accepted 3 July 2012 doi: 10.1111/j.1399-0039.2012.01941.x Abstract Current human leukocyte antigen (HLA) DNA typing methods such as the sequence- based typing (SBT) and sequence-specific oligonucleotide (SSO) methods generally yield ambiguous typing results because of oligonucleotide probe design limitations or phase ambiguity for HLA allele assignment. Here we describe the development and application of the super high-resolution single-molecule sequence-based typing (SS- SBT) of HLA loci at the 8-digit level using next generation sequencing (NGS). NGS which can determine an HLA allele sequence derived from a single DNA molecule is expected to solve the phase ambiguity problem. Eight classical HLA loci-specific polymerase chain reaction (PCR) primers were designed to amplify the entire gene sequences from the enhancer-promoter region to the 3 ′ untranslated region. Phase ambiguities of HLA-A, -B, -C, -DRB1 and -DQB1 were completely resolved and unequivocally assigned without ambiguity to single HLA alleles. Therefore, the SS- SBT method described here is a superior and effective HLA DNA typing method to efficiently detect new HLA alleles and null alleles without ambiguity. Introduction The major histocompatibility complex (MHC) region encodes the MHC transplantation and immuno-regulatory molecules that play important roles in the adaptive immune response. It is one of the medically important genomic regions that warrants special attention for genetic investigation because it has been implicated in numerous infectious and/or autoimmune diseases (1–3). The classical human MHC (human leukocyte antigen; HLA) loci, HLA-A, -B, -C, -DR, -DQ and -DP loci are distinguished by their extraordi- nary polymorphism, with over 6867 alleles implicated in ∗ These authors contributed equally to this work. disease resistance or susceptibility (IMGT/HLA Database release 3.7.0, http://www.ebi.ac.uk/imgt/hla/) (4). However, only 6.4% of the HLA alleles are determined from their entire gene sequence ranging from the enhancer–promoter region to the 3 ′ untranslated region (3 ′ UTR). Most alleles (93.6%) are defined by partial sequences of mainly exons 2 and 3 in HLA class I loci and mainly exon 2 in HLA class II loci (Table S1, Supporting Information ). The determination of HLA alleles by DNA typing tech- niques is necessary for HLA matching of donor and recip- ient at transplantation, medical research of HLA-related diseases and individual identification including paternity testing. More recently, HLA-typing is used also for preven- tion of drug adverse effects by in personalized medicine, © 2012 John Wiley & Sons A/S 305 Tissue Antigens, 2012, 80, 305–316