Identification of rare thalassemia variants using third-generation sequencing

Abstract

Routine PCR, Sanger sequencing, and specially designed GAP-PCR are often used in the genetic analysis of thalassemia, but all these methods have limitations. In this study, we evaluated a new third-generation sequencing-based approach termed comprehensive analysis of thalassemia alleles (CATSA) in subjects with no variants identified by routine PCR, Sanger sequencing, and specially designed GAP-PCR. Hemoglobin testing and routine PCR tests for 23 common variants were performed for 3,033 subjects. Then, Sanger sequencing and specially designed GAP-PCR were performed for a subject with no variants identified by routine PCR, no iron deficiency, and positive hemoglobin testing. Finally, the new CATSA method was conducted for the subjects with no variants identified by Sanger sequencing and specially designed GAP-PCR. In the 49 subjects tested by CATSA, eight subjects had variants identified. Sanger sequencing and independent PCR confirmed the CATSA result. In addition, it is the first time that Hb Lepore was identified in Hunan Province. In total, traditional methods identified variants in 759 of the 3,033 subjects, while CATSA identified additional variants in eight subjects. CATSA showed great advantages compared to the other genetic testing methods.

A Feasibility Study of Non-Invasive Prenatal Testing of Thalassemia by Haplotype Analysis Based on Next Generation Sequencing and Long-Read Sequencing

“…This is promising for NIPT. It has been reported that deletion and point mutation of HBA1, HBA2, and HBB can be directly obtained by TGS (Liu et al 2022;Liang et al 2021). Application of TGS for HBB in NIPT had been reported (Jiang et al 2021).…”

“…Recently, long-read thirdgeneration sequencing (TGS) has emerged as a powerful tool to detect the genetic basis of α-and β-thalassemia, especially for detecting complex structural variants and CNVs. TGS offers a convenient way to directly analyze parental haplotypes by utilizing ultra-long read lengths of up to 30 kb-100 kb, providing high accuracy (QV30 > 99.9%), single-molecule resolution, and no GC preference (Liang et al 2023;Liu 2022). This technology may improve the detection rate of NIPT in thalassemia.…”

Molecular Classifications in Gastric Cancer: A Call for Interdisciplinary Collaboration

“…Despite their potential, these techniques have not been integrated into clinical practice, and their utilization in research studies remains limited. Advantages over second-generation sequencing methods include the fact that they do not require sample pre-amplification and can read longer fragments of DNA, but the error rate is generally higher (10–15%) [ 58 , 59 , 60 , 61 ].…”

Third generation sequencing transforms the way of the screening and diagnosis of thalassemia: a mini-review

“…The findings suggest that rare thalassemia variants are more prevalent than previously thought and are often misdiagnosed by conventional diagnostic methods. Liu et al (29) conducted CATSA tests on 49 subjects who were negative for specially designed GAP-PCR and Sanger sequencing tests and identified mutations in the thalassemia gene in 8 subjects, indicating a higher positive detection rate of CATSA. The general process flow, advantages and disadvantages for screening and diagnosing thalassemia using conventional, NGS and TGS approaches.…”