Nanopore Third-Generation Sequencing for Comprehensive Analysis of Hemoglobinopathy Variants.

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.

One-step genotyping of α-thalassaemia by multiplex symmetric PCR melting curve.

AIMS: Alpha-thalassaemia is one of the most common monogenic disorders worldwide. Due to high guanine-cytosine (GC) content and high mutation diversity in α-globin gene cluster, deletional and non-deletional mutations were usually separately detected with different methods. The aim of this study was to develop a novel one-step method for α-thalassaemia genotyping. METHODS: A multiplex symmetric PCR melting curve strategy was designed for one-step α-thalassaemia genotyping. Based on this strategy, a novel method was developed to simultaneously detect four common deletional (-α3.7 , -α4.2 , _ _SEA , --THAI ) and five common non-deletional (αCD30(-GAG)α, αCD31(G>A)α, αWSα, αQSα, αCSα) α-thalassaemia mutations in a closed-tube reaction. This method was also evaluated by double-blind detection of 235 genotype-known samples and 1630 clinical samples. RESULTS: All nine α-thalassaemia mutations could be accurately identified by this novel method within 3 hours. The evaluation results also showed a 100% concordance with comparison methods. CONCLUSIONS: This method is rapid, accurate, low-cost and easy to operate, which can be used for molecular screening and genetic diagnosis of α-thalassaemia in clinical practice. The multiplex symmetric PCR melting curve strategy designed in this study can also provide an effective approach to the method development for high GC content templates and multiple mutations.