Third-generation sequencing: A novel tool detects complex variants in the α-thalassemia gene.

OBJECTIVE:Thalassemia is a monogenic disorder with a high carrier rate in the southern region of China. Most laboratories currently follow the protocol of testing hematologic indicators in individuals with positive hematologic indicators and then using the hot-spot mutation test kit. A novel thalassemia gene test is performed if there is a mismatch between the hematology and hot-spot mutation test results. However, due to the large population in southern China, some individuals carry complex α-globin gene cluster (CAGC) variants in NG_000006.1, which are difficult to detect using conventional thalassemia genetic analysis protocols, leading to missed or false genetic test results for individuals carrying these complex α-globin gene cluster variants. When an individual carries a complex α-thalassemia gene variant, and an individual carries a β- thalassemia gene variant, there may be clinical symptoms that might complicate clinical consultation and prenatal diagnosis if not accurately detected. Third-generation sequencing (TGS) enables long-read single-molecule sequencing with high detection accuracy, and long-length DNA chain reads in high-fidelity reads mode. TGS can be used to analyze high homology and rich GC DNA sequences. RESULTS:Four samples that showed abnormalities in the thalassemia genetic test were studied using TGS, revealing that they carried genotypes with complex α-globin gene cluster variants, one of which was a complex variant αα anti3.7 α anti3.7 α 17.2. CONCLUSIONS:TGS detects complex α-globin gene cluster variants. This study may provide a reference protocol for the use of TGS for the detection of complex α-globin gene cluster variants. TGS can reveal individuals with complex α-thalassemia genotypes in the population and improve the accuracy of genetic counseling and prenatal diagnosis.