三代测序与靶向捕获技术联用进行高分辨HLA基因分型及MHC区域单倍体型精细鉴定

doi:10.16288/j.yczz.18-282

遗传 ›› 2019, Vol. 41 ›› Issue (4): 337-348.doi: 10.16288/j.yczz.18-282

三代测序与靶向捕获技术联用进行高分辨HLA基因分型及MHC区域单倍体型精细鉴定

陈佳¹,舒明月¹,里进²,付爱思¹,杨帆³,王邹³,李一荣²(),邓子新¹(),刘天罡¹()

^1. 武汉大学药学院,组合生物合成与新药发现教育部重点实验室,武汉 430071
^2. 武汉大学中南医院检验医学中心,武汉 430071
^3. 武汉生物技术研究院公共技术服务平台,武汉 430071

收稿日期:2018-11-26 修回日期:2019-01-25 出版日期:2019-04-20 发布日期:2019-02-25
通讯作者: 李一荣,邓子新,刘天罡 E-mail:liyirong838@163.com;zxdeng@whu.edu.cn;liutg@whu.edu.cn
作者简介:陈佳,在读硕士研究生,专业方向：微生物与生化制药。E-mail: chenjia19940216@whu.edu.cn
基金资助:
“万人计划”青年拔尖人才项目资助

The third-generation sequencing combined with targeted capture technology for high-resolution HLA typing and MHC region haplotype identification

Chen Jia¹,Shu Mingyue¹,Li Jin²,Fu Aisi¹,Yang Fan³,Wang Zou³,Li Yirong²(),Deng Zixin¹(),Liu Tiangang¹()

^1. Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China
^2. Deparement of Laboratory,Zhongnan Hospital of Wuhan University, Wuhan 430071, China.
^3. Public Technology Service Platform, Wuhan Institute of Biotechnology, Wuhan 430071, China

Received:2018-11-26 Revised:2019-01-25 Online:2019-04-20 Published:2019-02-25
Contact: Li Yirong,Deng Zixin,Liu Tiangang E-mail:liyirong838@163.com;zxdeng@whu.edu.cn;liutg@whu.edu.cn
Supported by:
the Young Talents Program of National High-level Personnel of Special Support Program (The “Ten Thousand Talent Program”)

摘要/Abstract

摘要：

人类白细胞抗原(human leukocyte antigen,HLA)的高分辨率、精准分型对于组织配型以及HLA相关疾病研究具有重要意义。本研究以12位原发性肝细胞癌病人的外周血为供试样本,分析二、三代测序数据用于高分辨率HLA分型的优劣势,同时结合探针捕获与三代测序技术对YH、HeLa标准细胞系以及一个原发性肝细胞癌病人的主要组织相容性复合体(major histocompatibility complex,MHC)区域进行靶向分析,探究长读长测序技术对于整个MHC区域精细分析的潜力。研究表明：(1)二、三代测序技术均能实现6~8位高分辨HLA分型,且两者分型结果一致。但是三代数据的覆盖均一度显著优于二代,不会出现明显的“断层”现象;(2)超长的三代数据可直接跨越整个扩增子,对于基因单倍体型的判定(phasing)具有明显优势。样本中92.79%的HLA基因能够得到准确的单倍体分型结果,远高于二代的75.65%;(3)长读长的三代测序数据不但能实现对MHC区域的更好组装,还具有对整个MHC共计3.6 Mb区域进行phasing的能力,而这将有助于明确各个突变位点、等位基因、非编码区等基因原件在每个MHC单倍体型上的定位与相互连锁信息,为免疫等相关疾病的研究提供理论依据。

关键词: HLA, MHC, 三代测序, HLA单倍体分型, NimbleGen探针捕获技术

Abstract:

The high-resolution and accurate typing of human leukocyte antigen (HLA) is of great significance for the study of tissue matching in organ transplantation and the correlation between HLA and disease. In this study, the peripheral blood of 12 patients with primary hepatocellular carcinoma was used to compare the advantages and disadvantages of the next- and third-generation sequencing technology for high-resolution HLA typing. In addition, probe capture technology was used to capture the MHC region of YH and HeLa standard cell lines, and a primary hepatocellular carcinoma patient. The captured products were sequenced using PacBio platform to assess the potential of ultra-long reads sequencing technology for analysis of the entire MHC region. Our results showed that: (1) the next- and third-generation sequencing technology can both achieve 6-8 digit high resolution in HLA typing. However, the coverage of the third-generation is significantly better than the next-generation sequencing technology. (2) The ultra-long reads of the third generation sequencing can directly span the entire amplicon region, which has obvious advantages for haplotype phasing, with 92.79% of the HLA genes having accurate phasing results, which is much higher than the 75.65% from the next-generation data. (3) The long-reads from the third generating sequencing can not only be used to assemble the MHC region but also the ability to phase the entire MHC region of 3.6 Mb, thereby helping to clarify the localization information of the mutation sites, alleles and non-coding regions on each MHC haplotype, and providing a theoretical basis for the study of immune and other related diseases.

Key words: HLA, MHC, the third-generation sequencing, HLA haplotype phasing, NimbleGen probe capture technology

陈佳,舒明月,里进,付爱思,杨帆,王邹,李一荣,邓子新,刘天罡. 三代测序与靶向捕获技术联用进行高分辨HLA基因分型及MHC区域单倍体型精细鉴定[J]. 遗传, 2019, 41(4): 337-348.

Chen Jia,Shu Mingyue,Li Jin,Fu Aisi,Yang Fan,Wang Zou,Li Yirong,Deng Zixin,Liu Tiangang. The third-generation sequencing combined with targeted capture technology for high-resolution HLA typing and MHC region haplotype identification[J]. Hereditas(Beijing), 2019, 41(4): 337-348.

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三代测序与靶向捕获技术联用进行高分辨HLA基因分型及MHC区域单倍体型精细鉴定

The third-generation sequencing combined with targeted capture technology for high-resolution HLA typing and MHC region haplotype identification

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