Third-Generation Sequencing as a New Comprehensive Technology for Identifying Rare α- and β-Globin Gene Variants in Thalassemia Alleles in the Chinese Population

Abstract

Context.— Identification of rare thalassemia variants requires a combination of multiple diagnostic technologies. Objective.— To investigate a new approach of comprehensive analysis of thalassemia alleles based on third-generation sequencing (TGS) for identification of α- and β-globin gene variants. Design.— Enrolled in this study were 70 suspected carriers of rare thalassemia variants. Routine gap–polymerase chain reaction and DNA sequencing were used to detect rare thalassemia variants, and TGS technology was performed to identify α- and β-globin gene variants. Results.— Twenty-three cases that carried rare variants in α- and β-globin genes were identified by the routine detection methods. TGS technology yielded a 7.14% (5 of 70) increment of rare α- and β-globin gene variants as compared with the routine methods. Among them, the rare deletional genotype of –THAI was the most common variant. In addition, rare variants of CD15 (G>A) (HBA2:c.46G>A), CD117/118(+TCA) (HBA1:c.354_355insTCA), and β-thalassemia 3.5-kilobase gene deletion were first identified in Fujian Province, China; to the best of our knowledge, this is the second report in the Chinese population. Moreover, HBA1:c.-24C>G, IVS-II-55 (G>T) (HBA1:c.300+55G>T) and hemoglobin (Hb) Maranon (HBA2:c.94A>G) were first identified in the Chinese population. We also identified rare Hb variants of HbC, HbG-Honolulu, Hb Miyashiro, and HbG-Coushatta in this study. Conclusions.— TGS technology can effectively and accurately detect deletional and nondeletional thalassemia variants simultaneously in one experiment. Our study also demonstrated the application value of TGS-based comprehensive analysis of thalassemia alleles in the detection of rare thalassemia gene variants.

Molecular characterization of similar Hb Lepore Boston-Washington in four Chinese families using third generation sequencing

“…The third-generation sequencing based on PacBio Sequel II platform for globin genes variants detection was conducted as the description of our previous study 16 . Genomics DNA were extracted and then sent to Berry Genomics laboratory for third-generation sequencing.…”

“…After purification and end repair, double barcode adaptors were ligated to the 5’ and 3’ ends and Sequel Binding and Internal Ctrl Kit 3.0 (PacBio) was used to prepare SMRT bell libraries. Finally, third-generation sequencing was performed on the PacBio Sequel II System after primed DNA-polymerase complexes were loaded onto the SMRT cells 16 .…”

“…Cis- and trans-configuration between two variants in the long reads was analyzed using WhatsHap (version 0.18) software. The alignments of variant and wild-type molecules were visualized using the Integrative Genomics Viewer 16 .…”

“…At present, the third-generation sequencing (TGS) technology has been gradually used for thalassemia molecular diagnosis, with an obvious advantage in revealing globin genes single-nucleotide variants, short insertions/deletions and structural variants 13 , 14 . Increasing rare and novel globin gene variants have been identified using long-read sequencing 13 – 16 .…”

Identification of rare thalassemia variants using third-generation sequencing

“…In addition, TGS can detect both known and unknown variants and determine whether two or more variants are in cis- or trans-configurations ( Ardui et al, 2018 ; Liu et al, 2022 ). The clinical utility of a third-generation sequencing-based approach termed comprehensive analysis of thalassemia alleles (CATSA) in carrier screening of thalassemia was vigorously validated in blind clinical studies with a large cohort of samples, and CATSA demonstrated great advantages in terms of detecting range and accuracy compared to routine PCR and multiple ligation-dependent probe amplification ( Liang et al, 2021 ; Luo et al, 2022 ; Zhuang et al, 2022 ). In this study, the previous CATSA method was modified to cover more thalassemia variants.…”