Nanopore Third-Generation Sequencing for Comprehensive Analysis of Hemoglobinopathy Variants
Nanopore Third-Generation Sequencing for Comprehensive Analysis of Hemoglobinopathy Variants
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DOI：
10.1093/clinchem/hvad073
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发表时间：
2023-06-14
影响因子：
9.3
通讯作者：
Zhou，Wanjun
中科院分区：
文献类型：
--
作者：
Huang，Weilun;Qu，Shoufang;Zhou，Wanjun
多克斯福德纳米孔技术（ONT）第三代测序（TGS）是一个多功能的遗传诊断平台。然而，制备用于长读TGS的长模板文库仍然具有挑战性，特别是用于分析涉及复杂结构且发生在富含gc和/或同源区域的血红蛋白病变变异的ONT方法。方法设计多重长PCR制备文库模板，包括hba2 /1、HBG2/1、HBD、hbb全基因扩增子、靶向缺失和特殊结构变异等位基因扩增子。使用长pcr产物构建文库，并在Oxford Nanopore MinION仪器上进行测序。根据整合基因组学观察图（IGV）确定基因型。结果基于全基因序列的TGS方法区分了hba2 /1、HBG2/1、HBD和hbb的所有单核苷酸变异和结构变异。根据特异的等位基因读数，还鉴定出了靶向缺失和特殊的结构变异。158份α-/β-地中海贫血样本的结果与先前已知的基因型100%一致。结论该方法具有较高的通量，可用于血红蛋白病的分子筛选和遗传诊断。多重长PCR策略是一种高效的文库制备策略，为TGS检测开发提供了实用参考。
BackgroundOxford Nanopore Technology (ONT) third-generation sequencing (TGS) is a versatile genetic diagnostic platform. However, it is nonetheless challenging to prepare long-template libraries for long-read TGS, particularly the ONT method for analysis of hemoglobinopathy variants involving complex structures and occurring in GC-rich and/or homologous regions.MethodsA multiplex long PCR was designed to prepare library templates, including the whole-gene amplicons forHBA2/1,HBG2/1,HBD, andHBB, as well as the allelic amplicons for targeted deletions and special structural variations. Library construction was performed using long-PCR products, and sequencing was conducted on an Oxford Nanopore MinION instrument. Genotypes were identified based on integrative genomics viewer (IGV) plots.ResultsThis novel long-read TGS method distinguished all single nucleotide variants and structural variants withinHBA2/1, HBG2/1, HBD, andHBBbased on the whole-gene sequence reads. Targeted deletions and special structural variations were also identified according to the specific allelic reads. The result of 158 α-/β-thalassemia samples showed 100% concordance with previously known genotypes.ConclusionsThis ONT TGS method is high-throughput, which can be used for molecular screening and genetic diagnosis of hemoglobinopathies. The strategy of multiplex long PCR is an efficient strategy for library preparation, providing a practical reference for TGS assay development.