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

Gap- polymerase chain reaction (PCR), reverse dot-blot assay (RDB), real-time PCR based multicolor melting curve analysis (MMCA assay), multiplex ligation-dependent probe amplification (MLPA) and Sanger sequencing are conventional methods to diagnose thalassemia but all of them have limitations. In this study, we applied single-molecule real-time (SMRT) sequencing following multiplex long-range PCR to uncover rare mutations in nine patients and their family members. The patients with different results between Gap-PCR and MMCA assay or with phenotype not matching genotype were included. Using SMRT sequencing, we first identified the carriers with alpha alpha alpha(anti3.7)/HK alpha alpha, -alpha(762bp)alpha/alpha alpha (chr16:172,648-173,409), alpha alpha(fusion)/alpha(QS)alpha (in a trans configuration), two cases with novel gene rearrangements and another case with a novel 341 bp insertion in alpha-globin gene cluster, respectively. One carrier with -(SEA)/alpha alpha alpha(anti4.2), and two carriers with the coexistence of globin variant and an alpha-globin gene duplication were also found. Most importantly, we could determine two defects in alpha-globin gene cluster being a cis or trans configuration in a single test. Our results showed that SMRT has great advantages in detection of alpha-globin gene triplication, rare deletions and determination of a cis or trans configuration. SMRT is a comprehensive and one-step method for thalassemia screening and diagnosis, especially for detection of rare thalassemia mutations.