Method for establishing analysis immune library spectra based on random primer barcode technology

A technology of random primers and immune library, applied in the field of individualized diagnosis and treatment, can solve the problems of large sequencing error and amplification skew of low copy number sequences

Inactive Publication Date: 2013-12-11
ARMY MEDICAL UNIV
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AI Technical Summary

Problems solved by technology

[0006] In addition, the current high-throughput sequencing technology used for immune genome research has two defects: 1) amplification skew during library construction; 2) large sequencing errors for low copy number sequences

Method used

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Embodiment Construction

[0026] Preferred embodiments of the present invention will be described in detail below. The experimental method that does not indicate specific condition in the preferred embodiment, generally according to conventional conditions, such as Molecular Cloning Experiment Guideline (third edition, J. Sambrook etc., translated by Huang Peitang, etc., Science Press, 2002) described conditions, or as recommended by the manufacturer.

[0027] A sample selection of patients with chronic hepatitis B

[0028] (1) Select a patient with mild chronic hepatitis B: mild and few symptoms, such as mild fatigue, mild abdominal distension, mild liver discomfort, etc., and liver function damage. Transaminase is 3 times (normal value is 40 units), bilirubin is about 2 times normal value (normal value is 17.1 micromol / L), albumin is greater than or equal to 35 g / L (normal value is 35-55 g / L) Lift).

[0029] (2) Select one person with moderate chronic hepatitis B: the symptoms and signs and liver ...

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Abstract

The invention aims at the medical biotechnology, relates to a high-throughput sequencing method based on a primer barcode technology and an application thereof in study of immune genomics, and specifically relates to a T-cell high-throughput sequencing method based on a marking method of cDNA molecules. Specifically, the method comprises the following steps of extracting RNA from a sample, performing reverse transcription on the RNA to obtain cDNA, designing a specific primer according to the obtained cDNA, constituting a random primer barcode by a downstream primer of the designed specific primer, a random barcode and a sequencing joint in a series manner, adding excessive obtained random primer barcode in the obtained cDNA molecules, carrying out high-throughput sequencing by using the sequencing joint in the random primer barcode, carrying out VDJ comparison on sequencing data; analyzing a CDR3 sequence and establishing immune library spectra. The method reduces deflection and error generated in a PCR amplification process. The number of the cDNA molecules can be quantized according to the specific primer barcode corresponding to a given cDNA sequence.

Description

technical field [0001] The invention aims at the subject of medical biotechnology and belongs to the individualized diagnosis and treatment technology of major diseases, specifically a high-throughput sequencing method based on barcode technology and its application in immunogenomics research. Background technique [0002] Immunogenomics is the genomics of human major histocompatibility complex (MHC), T cell receptor (TCR) and antibodies, which can be widely used to guide vaccine design, individualized medicine and other research. In the study of immunogenomics, T cells are the executors of the body's immune function, and TCR is the molecule on the surface of T cells that recognizes antigens, and it is also the first key molecule for T cells to generate an immune response. During the development of T cells in the thymus, TCR through the rearrangement and selection of V(D)JC gene constitutes a peripheral diversity of T cell pool, so that it can respond to a variety of externa...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68
Inventor 尚小云吴玉章董惠王昊亮
Owner ARMY MEDICAL UNIV
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