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Method for establishing high-throughput sequencing library and application thereof

A sequencing library, high-throughput technology, applied in the biological field, can solve problems that need to be improved

Active Publication Date: 2013-05-15
BGI TECH SOLUTIONS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, the current research on the methylation detection of specific regions of the genome, such as promoter regions, CpG island regions, CpG extra-island regions, and imprinted gene regions, still needs to be improved.

Method used

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  • Method for establishing high-throughput sequencing library and application thereof
  • Method for establishing high-throughput sequencing library and application thereof
  • Method for establishing high-throughput sequencing library and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] In this embodiment, 2 μg of human peripheral blood mononuclear cell genomic DNA was used as a sample, and the implementation was carried out according to the following steps.

[0054] 1. Genomic DNA fragmentation:

[0055] Use the covaris-S2 fragmentation instrument to fragment the genomic DNA of the sample according to the parameters set in the table below, so as to obtain DNA fragments.

[0056] processing 1

Load ratio (%)

10

intensity

5

cycle / pulse

200

time(s)

50

processing 2

time(s)

0

processing 3

time(s)

0

Process 4

time(s)

0

cycle

3

[0057] The obtained DNA fragments are detected by electrophoresis, and the main band of the DNA fragments is required to be concentrated between 150-300bp, without protein and RNA contamination. Using QIAquick PCR Purification Kit (Qiagen) or magnetic bead purification, the DNA fragments th...

Embodiment 2

[0156] Using the Hiseq2000 sequencer, the high-throughput sequencing library of the specific genome region of the sample constructed in Example 1 was sequenced according to the read length of 90 bases at both ends, so as to obtain the sequencing results.

[0157] After the above-mentioned sequencing, the raw data is directly obtained, and the above-mentioned sequencing results can be obtained by performing basic analysis on the raw data, wherein the basic analysis process includes the following main steps: First, distinguish different samples through the sequence tags on the adapters or PCR primers library data; then, decontamination, joint removal, and low-quality filtering are performed on the raw data obtained by sequencing; finally, base conversion is performed on the previously processed data, specifically, all Cs of the positive strand are converted into Ts, All the Gs of the complementary strands were converted into A, thus, the sequencing results of the high-throughput ...

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Abstract

The invention discloses a method for establishing a high-throughput sequencing library and an application thereof. The method for establishing the high-throughput sequencing library comprises the following steps of: conducting DNA (Deoxyribose Nucleic Acid) fragmentation on a genome; conducting end repairing on DNA fragments; adding a basic group A to a 3' end of the DNA ends subjected to end repairing; connecting the DNA fragments with sticky ends A with methylated joints; conducting hybridization capture on the connection products by using a specific probe so as to obtain target fragments; conducting bisulfite treatment on the target fragments so as to convert non-methylated cytosine in the target fragments into uracil; conducting PCR (Polymerase Chain Reaction) amplification on the converted target fragments; separating and purifying the amplification products; and establishing the high-throughput sequencing library by using the amplification products. By utilizing the method for establishing the high-throughput sequencing library and the application thereof, the high-throughput sequencing library of a specific genome area of a sample can be conveniently and effectively established.

Description

technical field [0001] The present invention relates to the field of biotechnology. Specifically, it relates to DNA methylation detection technology, especially the methylation detection of a specific region of the genome. More specifically, the present invention provides a method for constructing a high-throughput sequencing library, a method for determining the methylation information of a specific genome region of a sample, and a method for determining the methylation information of a specific genome region of a sample. A device and a kit for constructing a high-throughput sequencing library of a specific genome region of a sample. Background technique [0002] DNA methylation is the most deeply studied epigenetic mechanism. DNA methylation plays an important role in maintaining normal cell function, inhibiting the damage of genome integrity caused by parasitic DNA components, modifying chromatin structure, inactivating X chromosome, genome imprinting, embryo It plays a...

Claims

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Application Information

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IPC IPC(8): C40B50/06C40B40/06C12N15/10C12Q1/68C12M1/34
CPCC40B40/08C40B50/06C12Q1/68C40B60/00C12N15/10C40B40/06C12N15/1093C12Q1/6806C12N15/11C12Q2523/125C12Q2525/117C12Q2525/191C12Q2535/122C12Q1/6876C12Q1/6869
Inventor 王君文高飞蒋慧武靖华吴红龙
Owner BGI TECH SOLUTIONS
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