Development Of A High-Throughput NGS Based Technique On Thalassemia-Gene Mutation And Deletion Screening

Thalassemia is a hereditary hemolytic anemia caused by globin gene deletion or point mutation.It is a single-gene genetic disease with the widest distribution and the largest cumulative population in the world.There is currently no effective cure for thalassemia,and the cost of treatment is high.Therefore,the birth of child with thalassemia,especially those of intermediate and severe thalassemia,imposes a heavy burden on family and social health care.Although it is difficult to cure,it can be prevented.Screening of thalassemia gene can be performed before and during pre-pregnancy.If the husband and wife screened positive for thalassemia,then prenatal diagnosis can be a measure that effectively prevent giving birth to a child with thalassemia.With the continuous improvement of PCR technology,gene detection technology has become the gold standard for the diagnosis of thalassemia,gradually replacing the traditional blood routine examination and hemoglobin electrophoresis technology.Clinical detection techniques of thalassemia include Gap-PCR,PCR-RDB,PCR flow fluorenseence immunmi crobeads assay,gene chip,MLPA,etc.These detection techniques have limitations,high cost or inability to simultaneously detect deletion and mutation.Therefore,it is urgent to develop a new technology with high throughput,low cost and simultaneous detection of deletion and mutation.In this study,a series of primers were designed based on the characteristics of ?-thalassemia and ?-thalassemia-related HBA1,HBA2 and HBB genes,and a index sequence for identifying samples was introduced.The multiplex PCR technique was used to amplify and enrich the HBA1,HBA2 and HBB genes in the sample.The PCR products of 96 samples were mixed into one library,and the library was constructed.The prepared library was sequenced on a high-throughput Hiseq2500 sequencing platform.The sequencing results of each DNA will be accurately mapped to each amplified region of each sample after being resolved by library adaptor sequence,sample index sequence and primer sequence alignment.The sequencing results of each sample and target region are compared with various types of thalassemia gene variants in the standard database to determine whether there are thalassemia gene mutations and the type of variation in the sample.The research results are as follows:(1)Successfully designed and verified 17 specific primers for the thalassemia-deficient typeand 10 primers for the thalassemia mutant,which can accurately amplify 7 kinds ofthalassemia-deficient types(–?3.7,–?4.2,--SEA,--THAI,SEA-HPFH,Chinese andTaiwanese)and all known thalassemia mutations in the HbVar database.(2)Successfully designed and verified 96 sets of primer labels,and the number of samples forone PCR reaction was upgraded from a traditional single sample to 96 samples,greatlyimproving the screening efficiency and cost savings of screening thalassemia.(3)Primer combinations,reaction systems and reaction procedures for multiplex PCRreactions were successfully established.Accurate and efficient amplification ofthalassemia mutant and deletion genes in 5 multiplex PCR reactions,compared with 11PCR reactions required for common PCR methodologies,this study simplifiesexperimental operation and saves screening costs.(4)In order to verify the accuracy and stability of the study,748 clinical samples(including452 positive samples and 290 negative samples)covering known results were selected forexperiments.710 samples were successfully genotyped,while 38 cases(5%)failed toobtain meaningful result.After comparison,the match rate of the 710 results was 100%,and the 38 cases of failed samples were re-examined to obtain accurate results of thethalassemia classification.Five commercially available reference samples(including 4positive and 1 negative)were selected and repeated 10 times.The results of 10 tests wereconsistent with the known results.Therefore,the high-throughput sequencing platformbased on the high-throughput sequencing platform developed in this study has thecharacteristics of high throughput,high accuracy and low cost,and is suitable for thescreening of thalassemia genes in large-scale large populations.