Nanopore Third-Generation Sequencing for Comprehensive Analysis of Hemoglobinopathy Variants

• Published in: Clinical chemistry (Baltimore, Md.) Md.), 2023-09, Vol.69 (9), p.1062-1071
• Main Authors: Huang, Weilun, Qu, Shoufang, Qin, Qiongzhen, Yang, Xu, Han, Wanqing, Lai, Yongli, Chen, Jiaqi, Zhou, Shihao, Yang, Xuexi, Zhou, Wanjun
• Format: Article
• Language: English
• ISSN: 0009-9147; 1530-8561
• DOI: 10.1093/clinchem/hvad073

Abstract Background Oxford 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. Methods A multiplex long PCR was designed to prepare library templates, including the whole-gene amplicons for HBA2/1, HBG2/1, HBD, and HBB, 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. Results This novel long-read TGS method distinguished all single nucleotide variants and structural variants within HBA2/1, HBG2/1, HBD, and HBB based 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. Conclusions This 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.