Comparison of Third-Generation Sequencing and Routine Polymerase Chain Reaction in Genetic Analysis of Thalassemia.

CONTEXT.—: Thalassemia is the most widely distributed monogenic autosomal recessive disorder in the world. Accurate genetic analysis of thalassemia is crucial for thalassemia prevention.

OBJECTIVE.—: To compare the clinical utility of a third-generation sequencing-based approach termed comprehensive analysis of thalassemia alleles with routine polymerase chain reaction (PCR) in genetic analysis of thalassemia and explore the molecular spectrum of thalassemia in Hunan Province.

DESIGN.—: Subjects in Hunan Province were recruited, and hematologic testing was performed. Five hundred four subjects positive on hemoglobin testing were then used as the cohort, and third-generation sequencing and routine PCR were used for genetic analysis.

RESULTS.—: Of the 504 subjects, 462 (91.67%) had the same results, whereas 42 (8.33%) exhibited discordant results between the 2 methods. Sanger sequencing and PCR testing confirmed the results of third-generation sequencing. In total, third-generation sequencing correctly detected 247 subjects with variants, whereas PCR identified 205, which showed an increase in detection of 20.49%. Moreover, α triplications were identified in 1.98% (10 of 504) hemoglobin testing-positive subjects in Hunan Province. Seven hemoglobin variants with potential pathogenicity were detected in 9 hemoglobin testing-positive subjects.

CONCLUSIONS.—: Third-generation sequencing is a more comprehensive, reliable, and efficient approach for genetic analysis of thalassemia than PCR, and allowed for a characterization of the thalassemia spectrum in Hunan Province.