Library building method for screening large samples of thalassemia based on high-throughput sequencing

Abstract

The invention discloses a library building method for screening large samples of thalassemia based on high-throughput sequencing. The library building method comprises the following steps: respectively amplifying HBA1, HBA2 and HBB genes by using specific primers with tag sequences, wherein the tag sequences are used for distinguishing different samples; mixing amplification products with same genes from different samples and then mixing the mixed amplification products with different genes; purifying the mixed products; incompletely interrupting the purified products; carrying out 5' phosphorylation blunt end repair and 3' end plus A to obtain a DNA segment with 5' phosphorylation and 3' viscous end A; ligating the DNA fragment with a connector containing a unique barcode sequence for distinguishing the library; purifying a ligation product to obtain an upper computer library suitable for the high-throughput sequencing. The method disclosed by the invention has the advantages that 245kinds of HBB gene mutation types and 93 kinds of HBA gene mutation types which are currently known and related to the thalassemia can be covered; meanwhile, novel types in the target area range can also be detected.

Description

technical field [0001] The invention relates to the technical field of thalassemia detection, in particular to a method for building a library based on high-throughput sequencing for screening large samples of thalassemia. Background technique [0002] Thalassemia is a thalassemia in which the synthesis of one or more globin chains in hemoglobin is absent or deficient due to a genetic defect. γ, δ, and β globin form the β gene family, and ζ and α globin form the α gene family; thalassemia is also divided into four types: α type, β type, δβ type, and δ type, among which β and α thalassemias are more common. common. The α-thalassemia-related genes include HBA2 and HBA1, and the β-thalassemia-related genes are HBB; there are as many as 348 types on HBA1, 431 mutation types on HBA2, and 887 mutation types on HBB; Among them, there are 484 mutation types related to thalassemia, including large deletions, point mutations, and deletion duplications. [0003] The occurrence of th...

AI Technical Summary

Problems solved by technology

Among them, the advantage of the sanger sequencing method is that it can detect known and unknown mutations in the target region, but its disadvantage is that the throughput is low and it is not suitable for large-scale screening

Chip hybridization and fluorescent quantitative PCR methods have relatively higher throughput, but only known mutations can be detected, and the detection sites are relatively small. During the large-scale screening of thalassemia, some rare mutations may be caused. missed detection

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