Nanopore Third-Generation Sequencing for Comprehensive Analysis of Hemoglobinopathy Variants.
Clin Chem
; 69(9): 1062-1071, 2023 09 01.
Article
en En
| MEDLINE
| ID: mdl-37311260
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-/ß-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.
Texto completo:
1
Base de datos:
MEDLINE
Asunto principal:
Nanoporos
/
Hemoglobinopatías
Tipo de estudio:
Diagnostic_studies
/
Prognostic_studies
Idioma:
En
Revista:
Clin Chem
Asunto de la revista:
QUIMICA CLINICA
Año:
2023
Tipo del documento:
Article