Detection of rare mutations in thalassemia using long-read single-molecule real-time sequencing

In this study, we applied the third generation sequencing following multiplex long PCR to detect nine thalassemia carriers and their family members with rare mutations. The assay included five primers for thalassemia. The results of genotypes for six patients were with the discrepancy between gap PCR and MMCA assay. One case with large deletion had normal MCV and MCH. Two samples with transfusion dependent thalassemia were only found one mutation in HBB gene. Using third generation sequencing, we identified the genotypes for these complex mutations. Most importantly, we confirmed that the repeating sequences in those samples were in a cis configuration. Two samples harbored a novel 341bp insertion which has never been reported. third generation sequencing has been identified to have great advantages in detection of globin gene triplications, large deletions and determining them in a cis or trans configuration.