Method for analyzing human blood group genotype based on high-throughput sequencing, and application thereof

A technology of sequencing analysis and genotyping, applied in the field of bioinformatics, can solve problems such as incomplete serological testing, insufficient antibody reagents, difficulties in diagnosis and treatment of blood transfusion and immune diseases, etc.

Inactive Publication Date: 2020-08-14
SHENZHEN BLOOD CENT
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the detection methods of the MNS blood group system are mainly serological methods, simple PCR-SSP method, generation sequencing method, DNA probe colorimetric detection and other methods, none of these methods can accurately identify the variation of the MNS blood group gene and the corresponding antigen , there are still many variant antigens that have not been confirmed. At the same time, due to the lack of antibody reagents of the MNS blood group system, various laboratories cannot complete the serological detection of this blood group, resulting in difficulties in the diagnosis and treatment of clinical blood transfusion and immune diseases.

Method used

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  • Method for analyzing human blood group genotype based on high-throughput sequencing, and application thereof
  • Method for analyzing human blood group genotype based on high-throughput sequencing, and application thereof
  • Method for analyzing human blood group genotype based on high-throughput sequencing, and application thereof

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Experimental program
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Embodiment 1

[0061] The technical route of this embodiment is as figure 1 shown. The specific implementation is as follows:

[0062] Randomly select 3,000 blood samples from voluntary blood donors and 175 representative samples and family investigation samples accumulated at ordinary times, and first use blood group serology to detect the type of MNS blood group antigen corresponding to red blood cell glycoprotein and the dose of red blood cell surface antigen (with Agglutination strength of antibody serum), select serologically representative samples to collect DNA for high-throughput whole genome sequencing.

[0063] Use high-throughput sequencing technology (NGS) to randomly fragment the genomic DNA of the selected sample, recover the DNA fragment of the required length (0.2-5kb) by electrophoresis, end repair - add adenylate deoxyribonucleotide, and connect the adapter , polymerase chain reaction, single-strand circularization, preparation of Nanoball-loading chip, using the method ...

s proportion 85

[0064] Obtain 30× whole genome sequencing (WGS) data, use SOAPnuke software to perform quality control analysis on off-machine data, filter low-quality Reads, obtain Q20 qualified Reads ratio of not less than 90%, and Q30 qualified Reads ratio of 85%. Focus on the analysis of GPA, GPB, GPE molecular corresponding GYPA, GYPB, GYPE gene and fusion gene GYP(A-B-A), GYP(B-A-B), GYP(A-B), GYP(A-B) The full-length sequence includes all information of exons, introns and UTR region sequences, such as image 3 shown. The inventors tested 30× whole genome sequencing data, the blood type gene detection rate was 100%, the average depth was 30.3×, and the average coverage rate was 100% for each gene. MNS blood group related genes GYPA / GYPB / GYPE The coverage rate is 98.3%, and the average depth is 25×, such as Figure 4 shown. It can meet the needs of research on the polymorphism of human blood group glycoprotein molecules, that is, the difficult blood group of MNS blood g...

Embodiment 2

[0069] Example 2 Cloud platform establishment

[0070] Such as Figure 9 As shown, the inventor has established a cloud platform for whole genome sequencing and blood typing, including an OSS cloud storage module, an ESC cloud service module and an RDS cloud data module.

[0071] The OSS cloud storage module unit, with a storage capacity of 10T, is used for sequencing data storage and can be used for long-term storage and reading and writing of data.

[0072] The ECS cloud data module is a cloud server equipped with 24-core 96GB running memory, 500GB high-efficiency SSD hard disk, and multiple genome-wide parallel computing capabilities to meet various biological information computing needs such as sequence comparison, mutation detection, and gene annotation.

[0073] The RDS cloud database will archive and build a database of the researched and newly released blood type genetic information, and establish a blood type information database.

[0074] The existing technology us...

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Abstract

The invention discloses a method for analyzing the human blood group genotype based on high-throughput sequencing, and belongs to the field of bioinformatics. In the method, first high-throughput sequencing data of human blood sample DNA are obtained, and further the sequencing data are processed through sequence comparison, mutation detection, and gene annotation to complete the analysis and verification of the blood group genotype. The invention establishes a whole gene sequencing blood typing cloud platform for the first time, the NGS technology is used to reveal the molecular mechanism ofhuman GPA, GPB, and GPE, complex GP (A-B-A), GP (B-A-B), and GP (A-B) fragments are sequenced and analyzed, polymorphism characteristics of glycoprotein heterozygous genes are analyzed in detail and the corresponding glycoprotein molecular type is determined through BWA / GATK and other bioinformatics software, mutant genes are overexpressed and an antigenic type of the MNS system is verified, and thereby difficult problems of clinical blood transfusion reactions and the diagnosis and treatment of immune diseases caused by heterozygous glycoprotein polymorphism molecules are solved.

Description

technical field [0001] The invention belongs to the field of bioinformatics, and in particular relates to a method for analyzing human blood genotypes based on high-throughput sequencing and its application. Background technique [0002] Human erythrocyte blood group glycoproteins: GPA, GPB and GPE all belong to erythrocyte transmembrane glycoproteins, and the corresponding coding genes are arranged on chromosome 4 in the order of 5'-GYPA-GYPB-GYPE-3', and the three have 95% identical Mutations such as exchange, fusion, recombination, and deletion during the genetic process will produce new antigenic determinants on the surface of red blood cells. The base sequences of the three genes determine the polymorphism of antigens in the human red blood cell MNS blood group system. The MNS blood group system is the second blood group system discovered by humans after the ABO blood group. MNS blood group is closely related to clinical blood transfusion and immune hemolytic diseases, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6869G16B30/00G16B20/20
CPCC12Q1/6888C12Q1/6869G16B30/00G16B20/20C12Q2600/156C12Q2535/122C12Q1/6881
Inventor 梁延连洪文旭齐彦伟徐筠娉史家海唐雄驰苏宇清吴凡梁爽
Owner SHENZHEN BLOOD CENT
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