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.
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.
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DOI：
10.3389/fimmu.2018.02294
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发表时间：
2018
影响因子：
7.3
通讯作者：
Shiina T
中科院分区：
文献类型：
--
作者：
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 JK;Shiina T
虽然NGS技术推动了用于HLA基因的鉴定和分类的高通量HLA基因分型方法的进步，以辅助疾病和移植中的精确医学工作，但这些方法的效率受到缺乏充分表征的高频率HLA等位基因参考序列数据库的阻碍。在这里，我们报告生产一个全面的收集全长HLA等位基因序列的8个经典的HLA基因座在日本人口中发现。我们用第三代SMRT测序方法提供的长扩增子跨越共识读段增强了Ion Torrent技术生成的第二代短读段数据，以创建在基因组编码和非编码水平解析的HLA I类和II类基因等位基因的参考级高质量序列。从以前用于建立日本受试者的HLA等位基因频率数据的参考组中获得46个DNA。这些样本包括在日本人群中集体等位基因频率超过99.2%的等位基因。使用先前设计的HLA基因座特异性引物通过长距离PCR独立扩增HLA基因座，随后使用SMRT和Ion PGM测序仪测序。在用于SMRT测序的参考感知软件工具LAA的帮助下，映射的长和短读段用于产生共有HLA等位基因序列的参考文库。共253个不同的等位基因被确定为46名健康受试者。其中，137个是新的等位基因：101个SNV和/或插入缺失和36个部分或全长水平的延伸等位基因。从核苷酸多样性的角度比较HLA序列显示，HLA-DRB 1是8个HLA基因中最分歧的，并且HLA-DPB 1基因序列分歧成两个不同的组，DP 2和DP 5，在外显子2中产生独立的多态性的证据。我们还确定了HLA-DRB 1中两个可能与类风湿性关节炎有关的特异性内含子变异。总之，通过第三代和第二代技术进行的全长HLA等位基因测序提供了基因组等位基因分辨率的多态性基因参考序列，包括分配到领域4水平的等位基因变异，为精准医学和HLA相关疾病和移植研究奠定了更坚实的基础。
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.
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影响因子：
7.3
作者：
Chitnis N;Clark PM;Kamoun M;Stolle C;Brad Johnson F;Monos DS
通讯作者：
Monos DS
影响因子：
10.6
作者：
Mahdi BM
通讯作者：
Mahdi BM
影响因子：
3.5
作者：
Hosomichi K;Shiina T;Tajima A;Inoue I
通讯作者：
Inoue I
影响因子：
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作者：
Dunn, D. S.;Inok, H.;Kulski, J. K.
通讯作者：
Kulski, J. K.
影响因子：
4.9
作者：
Gourraud, Pierre-Antoine;Dieude, Philippe;Constantin, Arnaud
通讯作者：
Constantin, Arnaud