Construction method and application of high-throughout sequencing library

A sequencing library, high-throughput technology, used in the methylation detection of specific regions of the genome, the construction of high-throughput sequencing libraries, the kit for high-throughput sequencing libraries in specific regions of the genome, and the field of DNA methylation detection. issues to be improved

Inactive Publication Date: 2014-05-21
BGI SHENZHEN CO LTD +1
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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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  • Construction method and application of high-throughout sequencing library
  • Construction method and application of high-throughout sequencing library

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] 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.

[0087] 1. Genomic DNA fragmentation:

[0088] 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.

[0089] processing 1

Load ratio (%)

10

[0090]

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

[0091] 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 qualifi...

Embodiment 2

[0189] 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.

[0190] 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 ...

Embodiment 3

[0203] Using Yanhuang cell line samples (Jun Wang et al.2008), repeat Example 1, except that the gene regions known to have methylation sites used to design specific probes are the codes of the genes listed in Table I Region and promoter region (867 genes in total after merging repeated genes), designed by eArray system, prepared by Agilent, the length of the probe is 12mer. Additionally, no bisulfite treatment step is required for resequencing and unmethylated sequencing libraries.

[0204] Using mixed index sequencing, the read length is 49bp, the index length is 6bp, the number of off-machine sequence fragments is 2.67Mb pairs, and the test data output is about 240Mb. Using the bwa alignment program, the sequenced fragments filtered for low-quality and contaminated adapters were aligned to the whole human genome. And a preliminary analysis of the comparison results was made.

[0205] Test results :

[0206] Table 3 shows the specific total amount of data off-machine of...

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Abstract

The invention discloses a construction method and an application of a high-throughout sequencing library. The construction method of the high-throughout sequencing library comprises the following steps: fragmenting genome DNA (deoxyribonucleic acid); repairing terminals of DNA fragments; adding a base A to 3' terminals of the DNA fragments with the repaired terminals; connecting the DNA fragments with sticky ends A to methylation linkers; carrying out hybrid capture on connecting products by using a specificity probe so as to obtain target fragments; carrying out bisulfite treatment on the target fragments so as to convert non-methylated cytosine in the target fragments to uracil; carrying out PCR (polymerase chain reaction) amplification on the converted target fragments; separating and purifying amplification products which form the high-throughout sequencing library. According to the method and application of the high-throughout sequencing library, the high-throughout sequencing library of a genome specific zone of a sample can be conveniently and efficiently constructed.

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): C40B50/06C12N15/11C12N15/10C12Q1/68C12M1/34
Inventor 高飞王童
Owner BGI SHENZHEN CO LTD
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