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

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Problems solved by technology

[0003] However, the current research on the methylation detection of specific regions of the genome, such as promo

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
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Example Embodiment

[0052] Example 1:

[0053] In this example, 2 μg of human peripheral blood mononuclear cell genomic DNA was used as a sample, and the following steps were performed.

[0054] 1. Genomic DNA fragmentation:

[0055] Use the covaris-S2 interrupter to fragment the sample genomic DNA according to the parameters set in the table below to obtain DNA fragments.

[0056] Treatment 1

[0057] The obtained DNA fragments are subjected to electrophoresis detection, and the main bands of the DNA fragments are required to be concentrated between 150-300bp, without protein and RNA contamination. Use QIAquick PCR purification kit (Qiagen) or magnetic beads to purify and re-dissolve the qualified DNA fragments into 32 μl elution buffer for use.

[0058] Use the same method to prepare 200-400ng fragmented λ-DNA, where λ-DNA is exogenous unmethylated.

[0059] 2. End repair:

[0060] 1) Prepare the end repair reaction system in a 1.5mL centrifuge tube according to the DNA fragment obtained in the previou...

Example Embodiment

[0155] Example 2:

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

[0157] After the above-mentioned sequencing, the original data is directly obtained. The above-mentioned sequencing results can be obtained by basic analysis of the original data. The basic analysis process includes the following main steps: Firstly, different samples are distinguished by the sequence tags on the adapters or PCR primers Then, the original data obtained by sequencing is decontaminated, jointed and filtered with low quality; finally, the data after the aforementioned processing is subjected to base conversion, specifically, all the C of the positive strand is converted into T, All the Gs of the complementary strands are converted into A, thereby obtaining the sequencing results of the high-throug...

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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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