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Method for constructing high-throughput sequencing library of immune repertoire

A technology of sequencing library and construction method, which is applied in the field of construction of high-throughput sequencing library of immune repertoire, and can solve problems such as errors

Inactive Publication Date: 2018-11-27
广州华银医学检验中心有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the library construction method based on 5'RACE also has corresponding disadvantages: errors may be introduced in the PCR step and on-machine sequencing process in library construction

Method used

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  • Method for constructing high-throughput sequencing library of immune repertoire
  • Method for constructing high-throughput sequencing library of immune repertoire
  • Method for constructing high-throughput sequencing library of immune repertoire

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0132] Example 1 Construction method of immune repertoire high-throughput sequencing library of micro-initial samples

[0133] Sample processing: Use lymphocyte separation medium to separate healthy human peripheral blood mononuclear cells and count them (2×10 6cells / ml), 20,000 cells were taken for subsequent BCR library construction experiments;

[0134] 1. Preparation of cDNA template

[0135] (1) Cell Lysis

[0136] Add 10000 lymphocytes to 100ul cell lysate for lysis to obtain lysate;

[0137] (2) mRNA isolation, reverse transcription, template replacement and UDG enzyme treatment

[0138] 1) Use the kit Dynabeads mRNA DIRECT Micro Kit (TermoFisher) to isolate mRNA from the lysed cell sample, and perform the operation according to the instructions;

[0139] 2) Configure the reverse transcription system as shown in Table 12, and immediately add it to the magnetic beads that have captured mRNA in the first step, resuspend the magnetic beads, and transfer them to PCR tub...

Embodiment 2

[0171] Example 2 The construction method of the high-throughput sequencing library of the immune repertoire of the conventional amount of initial samples

[0172] The sample in this embodiment takes the peripheral blood of a healthy person as a sample, and the specific implementation steps are as follows:

[0173] 1. Preparation of cDNA template

[0174] (1) Total RNA extraction

[0175] 1), lymphocyte separation;

[0176] 2), according to every 10 7 Add 1ml Trizol to each cell Add Trizol to the isolated lymphocytes to extract total RNA;

[0177] 3), the extracted RNA is subjected to agilent 2100 quality inspection;

[0178] (2) Reverse transcription, template replacement and UDG enzyme treatment (regular input sample)

[0179] 1), prepare the reaction system (mix1) shown in Table 14 in the PCR tube;

[0180] Table 18

[0181]

[0182] 2) Use a pipette or lightly flick the tube wall to mix the above reaction system, centrifuge briefly and place it on a PCR instrument...

Embodiment 3

[0217] By comparing the number of types of UMI before correction and the number of types of UMI after error correction to evaluate the impact on the sequencing accuracy, and multiple reads detected by the same UMI can be corrected each other to improve the accuracy of the reads sequence. The test uses 20 The data of each library is shown in Table 25:

[0218] Table 25

[0219]

[0220]

[0221] It can be seen that more than 70% of the UMI types have been corrected, indicating that the present invention has greatly improved the data accuracy. In addition, there are quite a few UMIs whose corresponding reads are greater than 2, and these reads can be mutually corrected , to remove PCR and sequencing errors and improve data accuracy.

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Abstract

The invention discloses a library construction scheme for introducing UM, namely molecular bar codes, based on a principle of 5'-RACE. The scheme specifically comprises the following steps: performingreverse transcription by designing single primers in a constant region, performing equivalent amplification to obtain all types of TCR / BCR CDR whole gene sequences, and introducing UMI, so that errors introduced in the subsequent PCR (Polymerase Chain Reaction) or sequencing process can be corrected. According to the method disclosed by the invention, the experimental requirements on library construction of trace samples as low as 100 cells can be met, and the TCR / BCR CDR whole gene sequences can be equivalently amplified.

Description

technical field [0001] The invention belongs to the field of biomedical services, and in particular relates to a method for constructing an immune repertoire high-throughput sequencing library. Background technique [0002] The immune repertoire is the sum of all functionally diverse B and T cells in the circulation of an individual at any given point in time. T cells and B cells respectively mediate the body's cellular and humoral immune responses, and recognize and bind antigens through their surface cell receptors (TCR or BCR), and then function to eliminate pathogens or tumor cells. [0003] A T or B lymphocyte expresses only one TCR or BCR, and each TCR or BCR consists of a variable region and a constant region. The constant regions of T and B cells in different clones can be the same, but the variable regions are different. Human T, BCR The total number of B cells is about 1012, so they have a complex diversity of antigen receptors. With the advent of next-generation...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/6806C12Q1/6869C40B50/06
CPCC12N15/1096C12Q1/6806C12Q1/6869C40B50/06C12Q2521/531C12Q2531/113C12Q2525/191C12Q2535/122
Inventor 李芬香李雪飞王勇斯董少玲王晓丹
Owner 广州华银医学检验中心有限公司
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