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首页> 美国卫生研究院文献>other >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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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

机译：全长HLA I和II类基因的多态性的参考等级表征在ION PGM系统上具有短读测序在PacBio平台上由单分子实时测序产生长读

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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.
机译：尽管NGS技术推动了高通量HLA基因分型方法的发展，这些方法可用于HLA基因的鉴定和分类，以协助疾病和移植方面的精准医学工作，但由于缺乏足够表征的高频HLA等位基因参考，这些方法的效率受到了阻碍。高度多态HLA基因系统的序列数据库。在这里，我们报道了为日本人群中发现的八个经典HLA基因座生成的全长HLA等位基因序列的全面集合。我们使用第三代SMRT测序方法提供的长扩增子，跨共识读段扩展了Ion Torrent技术生成的第二代短读数据，以创建在基因组上解析的参考级高质量HLA I类和II类基因等位基因序列编码和非编码级别。从先前用于在日本受试者中建立HLA等位基因频率数据的参考集中获得了46个DNA。样本中日本人群中具有集体等位基因频率的等位基因超过99.2％。使用先前设计的HLA-基因座特异性引物通过远程PCR独立扩增HLA基因座，然后使用SMRT和Ion PGM测序仪进行测序。借助用于SMRT测序的参考感知软件工具LAA，使用映射的长和短读数生成共有HLA等位基因序列的参考库。共为46名健康受试者确定了253个不同的等位基因。其中有137个是新颖的等位基因：101个SNV和/或indels和36个延伸的等位基因处于部分或全长水平。从核苷酸多样性的角度比较HLA序列发现，在8个HLA基因中HLA-DRB1差异最大，并且HLA-DPB1基因序列分为两个不同的组，即DP2和DP5，有证据表明在HLA中产生了独立的多态性。外显子2。我们还鉴定了HLA-DRB1的两个特定内含子变异，可能与类风湿关节炎有关。总之，通过第三代和第二代技术进行的全长HLA等位基因测序已以基因组等位基因分辨率提供了多态性基因参考序列，包括分配至field-4水平的等位基因变异，为精密医学和HLA-相关疾病和移植研究。

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