title: Cost-efficient multiplex PCR for routine genotyping of up to nine classical HLA loci in a single analytical run of multiple samples by next generation sequencing.
abstract: BACKGROUND
  HLA genotyping by next generation sequencing (NGS) requires three basic steps, PCR, NGS, and allele assignment. Compared to the conventional methods, such as PCR-sequence specific oligonucleotide primers (SSOP) and -sequence based typing (SBT), PCR-NGS is extremely labor intensive and time consuming. In order to simplify and accelerate the NGS-based HLA genotyping method for multiple DNA samples, we developed and evaluated four multiplex PCR methods for genotyping up to nine classical HLA loci including HLA-A, HLA-B, HLA-C, HLA-DRB1/3/4/5, HLA-DQB1, and HLA-DPB1.
  
  
  RESULTS
  We developed multiplex PCR methods using newly and previously designed middle ranged PCR primer sets for genotyping different combinations of HLA loci, (1) HLA-DRB1/3/4/5, (2) HLA-DQB1 (3.8 kb to 5.3 kb), (3) HLA-A, HLA-B, HLA-C, and (4) HLA-DPB1 (4.6 kb to 7.2 kb). The primer sets were designed to genotype polymorphic exons to the field 3 level or 6-digit typing. When we evaluated the PCR method for genotyping all nine HLA loci (9LOCI) using 46 Japanese reference subjects who represented a distribution of more than 99.5% of the HLA alleles at each of the nine HLA loci, all of the 276 alleles genotyped, except for HLA-DRB3/4/5 alleles, were consistent with known alleles assigned by the conventional methods together with relevant locus balance and no excessive allelic imbalance. One multiplex PCR method (9LOCI) was able to provide precise genotyping data even when only 1 ng of genomic DNA was used for the PCR as a sample template.
  
  
  CONCLUSIONS
  In this study, we have demonstrated that the multiplex PCR approach for NGS-based HLA genotyping could serve as an alternative routine HLA genotyping method, possibly replacing the conventional methods by providing an accelerated yet robust amplification step. The method also could provide significant merits for clinical applications with its ability to amplify lower quantity of samples and the cost-saving factors.
authors.affiliation: Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, Kanagawa, 259-1143, Japan. y-ozaki@tokai-u.jp.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Ozaki
authors.affiliation: Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, Kanagawa, 259-1143, Japan. shingo@tokai-u.jp.
authors.forename: Shingo
authors.initials: S
authors.lastname: Suzuki
authors.affiliation: HLA Laboratory, Japanese Red Cross Kanto-Koshinetsu Block Blood Center, Koto-ku, Tokyo, 135-8639, Japan. k-kashiwase@ktks.bbc.jrc.or.jp.
authors.forename: Koichi
authors.initials: K
authors.lastname: Kashiwase
authors.affiliation: Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, Kanagawa, 259-1143, Japan. ashige@is.icc.u-tokai.ac.jp.
authors.forename: Atsuko
authors.initials: A
authors.lastname: Shigenari
authors.affiliation: Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, Kanagawa, 259-1143, Japan. y-okuda@is.icc.u-tokai.ac.jp.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Okudaira
authors.affiliation: Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, Kanagawa, 259-1143, Japan. s-ito@tokai-u.jp.
authors.forename: Sayaka
authors.initials: S
authors.lastname: Ito
authors.affiliation: Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, Kanagawa, 259-1143, Japan. masuya@is.icc.u-tokai.ac.jp.
authors.forename: Anri
authors.initials: A
authors.lastname: Masuya
authors.affiliation: HLA Laboratory, Japanese Red Cross Kanto-Koshinetsu Block Blood Center, Koto-ku, Tokyo, 135-8639, Japan. f-azuma@ktks.bbc.jrc.or.jp.
authors.forename: Fumihiro
authors.initials: F
authors.lastname: Azuma
authors.affiliation: HLA Laboratory, Japanese Red Cross Kanto-Koshinetsu Block Blood Center, Koto-ku, Tokyo, 135-8639, Japan. t-yabe@ktks.bbc.jrc.or.jp.
authors.forename: Toshio
authors.initials: T
authors.lastname: Yabe
authors.affiliation: Department of Hematology, Fujita Health University School of Medicine, Toyoake, Aichi, 470-1192, Japan. smorishi@fujita-hu.ac.jp.
authors.forename: Satoko
authors.initials: S
authors.lastname: Morishima
authors.affiliation: Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, Kanagawa, 259-1143, Japan. s-mitsu@is.icc.u-tokai.ac.jp.
authors.forename: Shigeki
authors.initials: S
authors.lastname: Mitsunaga
authors.affiliation: HLA Laboratory, Japanese Red Cross Kanto-Koshinetsu Block Blood Center, Koto-ku, Tokyo, 135-8639, Japan. m-satake@ktks.bbc.jrc.or.jp.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Satake
authors.affiliation: Department of Legal Medicine, Shinshu University School of Medicine, Matsumoto, Nagano, 390-8621, Japan. otamasao@shinshu-u.ac.jp.
authors.forename: Masao
authors.initials: M
authors.lastname: Ota
authors.affiliation: Division of Epidemiology and Prevention, Aichi Cancer Center Research Institute, Nagoya, Aichi, 464-8681, Japan. ymorisim@aichi-cc.jp.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Morishima
authors.affiliation: Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, Kanagawa, 259-1143, Japan. kulski@me.com.|Centre for Forensic Science, The University of Western Australia, Nedlands, WA, 6008, Australia. kulski@me.com.
authors.forename: Jerzy K
authors.initials: JK
authors.lastname: Kulski
authors.affiliation: Research Department, One Lambda Inc, Part of Thermo Fisher Scientific, Kittridge Street, Canoga Park, CA, 91303-2801, USA. Yuki.Saito@thermofisher.com.
authors.forename: Katsuyuki
authors.initials: K
authors.lastname: Saito
authors.affiliation: Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, Kanagawa, 259-1143, Japan. hinoko@is.icc.u-tokai.ac.jp.
authors.forename: Hidetoshi
authors.initials: H
authors.lastname: Inoko
authors.affiliation: Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, Kanagawa, 259-1143, Japan. tshiina@is.icc.u-tokai.ac.jp.
authors.forename: Takashi
authors.initials: T
authors.lastname: Shiina
mesh_terms: D000483:Alleles; D017931:DNA Primers / Q000378:metabolism; D056426:Genetic Loci; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods*; D006680:HLA Antigens / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D006680:HLA Antigens