A next generation sequencing based universal target panel and algorithm for one stop detection of copy number alterations and single nucleotide variations in the HBB gene cluster for rapid diagnosis of β-Thalassemia

Abstract Background This study aimed to develop and validate a targeted next-generation sequencing (NGS) panel along with a data analysis algorithm to detect single nucleotide variants (SNVs) and copy number variations (CNVs) within the beta-globin gene cluster. The goal was to reduce turnaround time (TAT) compared to conventional genotyping methods and provide a rapid, comprehensive solution for prenatal diagnosis, carrier screening, and genotyping of β-thalassemia cases. Methods and Results We designed a targeted NGS panel covering an 80.4 kb region on chromosome 11, including the beta-globin gene cluster and the 5' locus control region (LCR). An advanced data analysis algorithm was developed, integrating variant calling and depth plot analysis, to enable the simultaneous detection of SNVs and CNVs in a single run. The panel and algorithm were validated using 14 in-house β-thalassemia unique carrier/patient samples and annotated variants reported in HbVar database. We identified seven pathogenic SNVs and five CNVs in the beta-globin gene cluster across various genetic conditions, including heterozygous, homozygous, and compound heterozygous states. Through HbVar database, additionally, we assessed 169 rare deletions and 11 fusion mutations reported in the HbVar database to verify the theoretical capability of our panel to detect all CNVs within the target region. Conclusion The developed NGS panel and algorithm effectively detect both SNVs and CNVs in a single run and can be applied for prenatal diagnosis and carrier screening of hemoglobinopathies, demonstrating its versatility and clinical utility.