Establishment And Application Of A New Method To Detect Thalassemia Using Combined Probe-anchored Polymer Sequencing

There is a high carrying rate of thalassemia in coastal areas such as Southeast Asia and the Mediterranean.It is currently the world's highest incidence of genetic hemolytic disease.The pathogenic principle is hereditary anemia caused by single or complex structural abnormalities or synthetic abnormalities of ? or ? globin chains.At the gene level,it is due to mutations or deletions of genes those encoding alpha ? and ? globin(HBA1,HBA2,and HBB),which can be detected and confirmed by sequencing.The NGS method,which based on sequencing by synthesis with low cost and high detection throughput,is currently the most widely used.While the mainstream NGS platforms on the market,such as Hiseq and Miseq serial sequencing instruments and supporting reagents,are subject to foreign manufacturers or policies.Their clinical applications are not conducive to the screening of thalassemia.This paper mainly studies the application of a domestically developed NGS platform MGISEQ-2000,which based on the combined probe anchored polymer sequencing technology in the detection of thalassemia genes.Based on the general purpose fragment amplification and library construction methods,329 cases of thalassemia samples with known results were used for research and development,the primers and experimental detection procedures were optimized,and information analysis methods were established.Finally,this method was used to test 2000 samples tested by Hiseq-2500 platform,and the performance of this method was analyzed.The research results are as follows:A series of primers for amplifying the target gene fragments of thalassemia mutants and deletions were redesigned through the comparative study of various variant sequences of thalassemia genes,and the target gene fragments were confirmed by agarose gel electrophoresis.The size was consistent with expectations,and the accuracy of the designed primers was verified by Sanger sequencing.After amplifying and building the 329 samples with known results,the method of separating nucleic acid fragments and the combined anchor probe sequencing method were used to filter and analyze the data from the machine.The results shows that nucleic acid fragments of different sizes were separated,which can achieve the relative homogenization of the copy number of the fragments of different DNB sizes,the sequencing signal strength is relatively consistent,improve the uniformity of data distribution,and the depth coverage of the target area is better,the positive reference value for information analysis is established,thus determining the platform Thalassaemia genetic testing technology.Using combined anchor probe sequencing technology to detect and analyze thalassemia genes of 2000 samples,the results are compared with the detection of Hiseq platform,the two test methods have the same test results for 2000 thalassemia screening samples,various types The thalassemia variants can be detected,and various types of positive samples are selected for verification by Sanger and Gap-PCR.The results are in line with expectations,further proving the feasibility of the method from the side.In summary,the thalassemia gene detection technology based on the combined probe anchored polymer sequencing technology is suitable for thalassemia gene detection.Nucleic acid fragment separation technology can provide a new detection method for other direct library-building methods based on PCR amplification products.This system belongs to the domestically-developed domestic sequencing system.It has the advantages of high throughput,high accuracy,and controllable cost.It has broad clinical application prospects and is suitable for laboratory testing of large sample sizes.