A New Method To Detect α-thalassemia Deletions Using Nextgeneration Sequencing

Thalassemia is one of the most common hereditary disease worldwide, easily found in Mediterranean, the Middle East, India, Southeast Asia and Africa. Thalassemia also very common in southern China, it is a major health threat to local people. Thalassemia is caused by gene mutations in the DNA of cells that make hemoglobin, the mutations that cause thalassemia disrupt the normal production of hemoglobin and cause low hemoglobin levels and a high rate of red blood cell destruction. The several types of thalassemia may be divided into two groups,α and β, according to mutation types of the globin gene. α-thalassemia mainly cause by deletion or SNV(single nucleotide variation) in hemoglobin gene, because of the deletion types are more often. The Thalassemia prevention and control is an important part of Chinese birth defects control policy, targeted screening in premarital and Pre-pregnancy of this genetic disorders are very efficient. So that to develop more efficient hemoglobin gene test method is very important and will be helpful to the disease control.Gap-PCR and Real-Time PCR is commonly used to detect α-thalassemia deletion. But more unknown deletions can’t be detected by Gap-PCR, more than 2-3 repeat each sample in the experiment by Real-Time PCR and the fluorescent signal fluctuation will affect the judgement. In this study, α-globin gene was studied in this paper, According to the defects of existing genetic testing technology for α-thalassemia. We develop a new genetic technology for α thalassemia based on next generation sequencing. Primerindex was used in PCR amplification, most of the long fragment deletion common area in α-globin was amplified, and which cover part of HBA1 and HBA2 genes. Through sequencing library building for the PCR amplification and combined with the adaptor sequencing on next generation sequencing, sequencing was completed on Illumina Hiseq2000 platform. In this study, relative quantitative was used in results analysis,according to the sequencing reads depth in each gene. In order to verify the feasibility of this study, we select 950(120 positive and 830 negative)samples with known results to validate this method, the coincidence rate was 97.1%. Through the supplementary experiment, the result shows that α-globin long deletion and gene copy number increase can accurately detected by this new method. We have developed a new detecting technique for deletion types of α-thalassemia, the technique base on NGS sequencing technology platform which is marked high throughput, lower cost and high accuracy. It’s quite sure if this new technique translate into clinical use that will benefit thalassemia screening and control a lot.