Gao Su-Qing, Quan Zhan-Rou, Zhong Yan-Ping, Chen Hao, He Liu-Mei, Zou Hong-Yan, Deng Zhi-Hui
Institute of Transfusion Medicine, Shenzhen Blood Center, Shenzhen 518025, Guangdong Province, China.E-mail:
Institute of Transfusion Medicine, Shenzhen Blood Center, Shenzhen 518025, Guangdong Province, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2024 Apr;32(2):603-609. doi: 10.19746/j.cnki.issn.1009-2137.2024.02.042.
To investigate the accuracy of next-generation sequencing technology (NGS) in detecting the polymorphisms of and alleles in randomly-selected unrelated healthy individuals from Shenzhen Han population, investigate the potential reason for allele dropout in routine NGS, and establish an internal quality control system.
NGS-based HLA class II genotyping was performed on 1 012 samples using the MiSeqDx platform. The suspected missed alleles indicated by the quality control software and homozygotes were confirmed by PCR-SSOP or PCR-SBT methods.
A total of 139 alleles were detected, including (45), (7), (5), (7), (17), (21), (10) and (27). *09:01(17.09%),15:01(10.72%); *02:02(25.99%),03:01(10.18%); *01:03(36.46%); *01:01(15.42%); *01:02(20.01%),03:02(17.19%); *03:01(19.47%),03:03(17.98%), 05:02(11.66%), 06:01(10.67%); *02:02(54.45%), 01:03(31.18%) and *05:01(39.13%), 02:01(16.90%) alleles were the most common alleles in Shenzhen Han population (frequencies >10%). There was no statistical difference between the gene frequencies of and loci in our study. The HLA Common and Well-Documented Alleles in China (CWD2.4) (χ=12.68, >0.05). 94 cases of homozygous samples detected by NGS were retested by PCR-SSOP or SBT method, and one case of allele dropout at locus was found. SBT method confirmed that the allele of *04:03 was missed. The laboratory internal quality control system was established. Two cases of new alleles were detected and named by WHO Nomenclature Committee for Factors of the HLA System.
The HLA genotyping results based on NGS showed a significantly lower ambiguity rate. The HLA class II alleles exhibit genetic polymorphism in the Han population of unrelated healthy individuals in Shenzhen. The independent method based on NGS in clinical histocompatibility testing has limitations and requires internal quality control strategies to avoid allele-dropout events.
探讨新一代测序技术（NGS）检测深圳汉族随机选取的无血缘关系健康个体中 及 等位基因多态性的准确性，研究常规NGS中等位基因缺失的潜在原因，并建立内部质量控制体系。
使用MiSeqDx平台对1012份样本进行基于NGS的HLA II类基因分型。通过PCR-SSOP或PCR-SBT方法对质量控制软件提示的可疑漏检等位基因和 纯合子进行确认。
共检测到139个等位基因，包括（45）、（7）、（5）、（7）、（17）、（21）、（10）和（27）。*09:01（17.09%）、15:01（10.72%）；*02:02（25.99%）、03:01（10.18%）；*01:03（36.46%）；*01:01（15.42%）；*01:02（20.01%）、03:02（17.19%）；03:01（19.47%）、03:03（17.98%）、05:02（11.66%）、06:01（10.67%）；02:02（54.45%）、01:03（31.18%）和05:01（39.13%）、02:01（16.90%）等位基因是深圳汉族人群中最常见的等位基因（频率>10%）。本研究中 及 位点的基因频率无统计学差异。中国HLA常见及充分记录等位基因（CWD2.4）（χ=12.68，>0.05）。对NGS检测出的94例 纯合子样本采用PCR-SSOP或SBT方法进行复检，发现1例 位点等位基因缺失。SBT方法确认04:03等位基因漏检。建立了实验室内部质量控制体系。检测到2个新等位基因并由世界卫生组织HLA系统因子命名委员会命名。
基于NGS的HLA基因分型结果显示模糊率显著降低。深圳汉族无血缘关系健康个体中HLA II类等位基因存在遗传多态性。临床组织相容性检测中基于NGS的独立方法存在局限性，需要内部质量控制策略以避免等位基因缺失事件。
