Application Of Next Generation Sequencing To Detect The Thalassemia Genes

Objective: By comparing the gene detected results of next-generation sequencing and traditional hematological methods(Gap-PCR and PCR-RDB technology),to distinguish the differencies both varecity and sensitivity of two methods.Further evaluate the feasibility of adopt next-generation sequencing to test thalassemia gene in high-prevalence regions of thalassemia.to establish an new process which detect thalasemia gene mutation in high-prevalence regions of thalassemia based on next-generation sequencing.Methods: This study was been divided into two phases.Stage one: randomly collected 900 clinical cases of positive hematology test from January 2015 to December 2016 in the Prenatal diagnosis center of our hospital.Extraction of peripheral venous blood 2~3ml,EDTA anticoagulant and specimens were conducted with next-generation sequencing technology and traditional thalassemia gene detection method to test the thalassemia genes,with which the experiment results were compared and analyzed.Stage two: useing next-generation sequencing technology to detect the thalassemia gene of thalassemia gene carrier’s mate.These thalassemia carriers were detected on stage one.meanwhile,routine blood examinations and hemoglobin electrophoresis were been tested.then analyze examination results.Results: In stage one,among 900 tested individuals,585 were detected with thalassemia gene mutation carriers by using traditional thalassemia gene detection method,which were screened out with positive rate as 65.00%(585/900).Among 585 thalassemia carriers,326 of them were α-thalassemia,or 55.73%(326/585),244 of them were β-thalassemia,or 41.54%(244/585),the proportion of αβ-thalassemia is 2.56 %(15/585).Compared with the traditional thalassemia gene detection method,next-generation sequencing detected 605 cases with thalassemia genetic abnormalities.The positive rate of 900 people were 67.22%,which including α-thalassaemia gene mutation in 333 cases,the positive ratio is 55.04%(333/605),β-thalassemia gene mutation detectioon rates were 41.81%(253/585).Conposite α and β-thalassemia gene mutation rates were 3.14%(19/605).Through contrastive analysis proved that positive rates detceted by next-generation sequencing is higher than traditional thalassemia test method(p<0.01).In stage two,64 thalassemia gene mutation carries were identified from 485 samples in total.More than 71.87%(46/64)were α-thalassemia gene mutation,25% were β-thalassemia gene mutation,conposite α and β-thalassemia gene carrier have 2 cases.Among 64 thalassemia gene mutation samples,21 whose routine blood examinations and hemoglobin electrophoresis were normal,this means that if using traditional approaches,21 thalassemia carriers will been missed.The missed rates reach up to 32.81%(21/64).The thalassemia gene mutation cases that detected by next-generation sequencing technologies including 20 rare types or New mutations,in total 25 cases.Parts of genotype have not been reported all over the country even in the worldwide.Conclusion: Next-generation sequencing technology is superior to the traditional thalassemia gene detection method.The results showed that using next-generation sequencing technology to screening thalassemia gene have clinical feasibility.Next-generation sequencing technology is deserved to be popularized especially in high-prevalence regions of thalassemia.