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Reference Grade Characterization of Polymorphisms in Full-Length HLA Class I and II Genes With Short-Read Sequencing on the ION PGM System and Long-Reads Generated by Single Molecule, Real-Time Sequencing on the PacBio Platform.
Front Immunol. 2018; 9:2294.FI

Abstract

Although NGS technologies fuel advances in high-throughput HLA genotyping methods for identification and classification of HLA genes to assist with precision medicine efforts in disease and transplantation, the efficiency of these methods are impeded by the absence of adequately-characterized high-frequency HLA allele reference sequence databases for the highly polymorphic HLA gene system. Here, we report on producing a comprehensive collection of full-length HLA allele sequences for eight classical HLA loci found in the Japanese population. We augmented the second-generation short read data generated by the Ion Torrent technology with long amplicon spanning consensus reads delivered by the third-generation SMRT sequencing method to create reference grade high-quality sequences of HLA class I and II gene alleles resolved at the genomic coding and non-coding level. Forty-six DNAs were obtained from a reference set used previously to establish the HLA allele frequency data in Japanese subjects. The samples included alleles with a collective allele frequency in the Japanese population of more than 99.2%. The HLA loci were independently amplified by long-range PCR using previously designed HLA-locus specific primers and subsequently sequenced using SMRT and Ion PGM sequencers. The mapped long and short-reads were used to produce a reference library of consensus HLA allelic sequences with the help of the reference-aware software tool LAA for SMRT Sequencing. A total of 253 distinct alleles were determined for 46 healthy subjects. Of them, 137 were novel alleles: 101 SNVs and/or indels and 36 extended alleles at a partial or full-length level. Comparing the HLA sequences from the perspective of nucleotide diversity revealed that HLA-DRB1 was the most divergent among the eight HLA genes, and that the HLA-DPB1 gene sequences diverged into two distinct groups, DP2 and DP5, with evidence of independent polymorphisms generated in exon 2. We also identified two specific intronic variations in HLA-DRB1 that might be involved in rheumatoid arthritis. In conclusion, full-length HLA allele sequencing by third-generation and second-generation technologies has provided polymorphic gene reference sequences at a genomic allelic resolution including allelic variations assigned up to the field-4 level for a stronger foundation in precision medicine and HLA-related disease and transplantation studies.

Authors+Show Affiliations

Division of Basic Medical Science and Molecular Medicine, Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Japan.Molecular Biology Applications, Pacific Biosciences, Inc, Menlo Park, CA, United States.Pacific Biosciences Division, Tomy Digital Biology Co., Ltd, Tokyo, Japan.Division of Basic Medical Science and Molecular Medicine, Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Japan.Division of Basic Medical Science and Molecular Medicine, Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Japan.Division of Basic Medical Science and Molecular Medicine, Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Japan.The Institute of Medical Sciences, Tokai University, Isehara, Japan.GenoDive Pharma Inc., Atsugi, Japan.Molecular Biology Applications, Pacific Biosciences, Inc, Menlo Park, CA, United States.Molecular Biology Applications, Pacific Biosciences, Inc, Menlo Park, CA, United States.Pacific Biosciences Division, Tomy Digital Biology Co., Ltd, Tokyo, Japan.Pacific Biosciences Division, Tomy Digital Biology Co., Ltd, Tokyo, Japan.Division of Endocrinology, Diabetes, and Metabolism, Hematology, Rheumatology (Second Department of Internal Medicine), Graduate School of Medicine, University of the Ryukyus, Nishihara, Japan.Department of Promotion for Blood and Marrow Transplantation, Aichi Medical University School of Medicine, Nagakute, Japan.GenoDive Pharma Inc., Atsugi, Japan.Division of Basic Medical Science and Molecular Medicine, Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Japan. School of Psychiatry and Clinical Neurosciences, The University of Western Australia, Perth, WA, Australia.Division of Basic Medical Science and Molecular Medicine, Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Japan.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

30337930

Citation

Suzuki, Shingo, et al. "Reference Grade Characterization of Polymorphisms in Full-Length HLA Class I and II Genes With Short-Read Sequencing On the ION PGM System and Long-Reads Generated By Single Molecule, Real-Time Sequencing On the PacBio Platform." Frontiers in Immunology, vol. 9, 2018, p. 2294.
Suzuki S, Ranade S, Osaki K, et al. Reference Grade Characterization of Polymorphisms in Full-Length HLA Class I and II Genes With Short-Read Sequencing on the ION PGM System and Long-Reads Generated by Single Molecule, Real-Time Sequencing on the PacBio Platform. Front Immunol. 2018;9:2294.
Suzuki, S., Ranade, S., Osaki, K., Ito, S., Shigenari, A., Ohnuki, Y., Oka, A., Masuya, A., Harting, J., Baybayan, P., Kitazume, M., Sunaga, J., Morishima, S., Morishima, Y., Inoko, H., Kulski, J. K., & Shiina, T. (2018). Reference Grade Characterization of Polymorphisms in Full-Length HLA Class I and II Genes With Short-Read Sequencing on the ION PGM System and Long-Reads Generated by Single Molecule, Real-Time Sequencing on the PacBio Platform. Frontiers in Immunology, 9, 2294. https://doi.org/10.3389/fimmu.2018.02294
Suzuki S, et al. Reference Grade Characterization of Polymorphisms in Full-Length HLA Class I and II Genes With Short-Read Sequencing On the ION PGM System and Long-Reads Generated By Single Molecule, Real-Time Sequencing On the PacBio Platform. Front Immunol. 2018;9:2294. PubMed PMID: 30337930.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Reference Grade Characterization of Polymorphisms in Full-Length HLA Class I and II Genes With Short-Read Sequencing on the ION PGM System and Long-Reads Generated by Single Molecule, Real-Time Sequencing on the PacBio Platform. AU - Suzuki,Shingo, AU - Ranade,Swati, AU - Osaki,Ken, AU - Ito,Sayaka, AU - Shigenari,Atsuko, AU - Ohnuki,Yuko, AU - Oka,Akira, AU - Masuya,Anri, AU - Harting,John, AU - Baybayan,Primo, AU - Kitazume,Miwako, AU - Sunaga,Junichi, AU - Morishima,Satoko, AU - Morishima,Yasuo, AU - Inoko,Hidetoshi, AU - Kulski,Jerzy K, AU - Shiina,Takashi, Y1 - 2018/10/04/ PY - 2018/04/16/received PY - 2018/09/17/accepted PY - 2018/10/20/entrez PY - 2018/10/20/pubmed PY - 2019/10/8/medline KW - HLA KW - Ion PGM KW - NGS KW - PacBio RS II KW - SMRT sequencing KW - genotyping KW - human leukocyte antigen KW - next-generation sequencing SP - 2294 EP - 2294 JF - Frontiers in immunology JO - Front Immunol VL - 9 N2 - Although NGS technologies fuel advances in high-throughput HLA genotyping methods for identification and classification of HLA genes to assist with precision medicine efforts in disease and transplantation, the efficiency of these methods are impeded by the absence of adequately-characterized high-frequency HLA allele reference sequence databases for the highly polymorphic HLA gene system. Here, we report on producing a comprehensive collection of full-length HLA allele sequences for eight classical HLA loci found in the Japanese population. We augmented the second-generation short read data generated by the Ion Torrent technology with long amplicon spanning consensus reads delivered by the third-generation SMRT sequencing method to create reference grade high-quality sequences of HLA class I and II gene alleles resolved at the genomic coding and non-coding level. Forty-six DNAs were obtained from a reference set used previously to establish the HLA allele frequency data in Japanese subjects. The samples included alleles with a collective allele frequency in the Japanese population of more than 99.2%. The HLA loci were independently amplified by long-range PCR using previously designed HLA-locus specific primers and subsequently sequenced using SMRT and Ion PGM sequencers. The mapped long and short-reads were used to produce a reference library of consensus HLA allelic sequences with the help of the reference-aware software tool LAA for SMRT Sequencing. A total of 253 distinct alleles were determined for 46 healthy subjects. Of them, 137 were novel alleles: 101 SNVs and/or indels and 36 extended alleles at a partial or full-length level. Comparing the HLA sequences from the perspective of nucleotide diversity revealed that HLA-DRB1 was the most divergent among the eight HLA genes, and that the HLA-DPB1 gene sequences diverged into two distinct groups, DP2 and DP5, with evidence of independent polymorphisms generated in exon 2. We also identified two specific intronic variations in HLA-DRB1 that might be involved in rheumatoid arthritis. In conclusion, full-length HLA allele sequencing by third-generation and second-generation technologies has provided polymorphic gene reference sequences at a genomic allelic resolution including allelic variations assigned up to the field-4 level for a stronger foundation in precision medicine and HLA-related disease and transplantation studies. SN - 1664-3224 UR - https://www.unboundmedicine.com/medline/citation/30337930/Reference_Grade_Characterization_of_Polymorphisms_in_Full_Length_HLA_Class_I_and_II_Genes_With_Short_Read_Sequencing_on_the_ION_PGM_System_and_Long_Reads_Generated_by_Single_Molecule_Real_Time_Sequencing_on_the_PacBio_Platform_ L2 - https://doi.org/10.3389/fimmu.2018.02294 DB - PRIME DP - Unbound Medicine ER -