Genome methylation library, preparation method and application thereof

A methylation and genome technology, applied in the field of genome methylation library and its preparation, can solve the problems of base imbalance, reduce the comparison rate of reads containing non-methylated cytosine, and cannot completely cover the CpG region, etc. Achieve the effect of improving the quality of sequencing and improving the base balance of the library

Pending Publication Date: 2021-11-19
GUANGZHOU DRIPNANO BIOTECH CO LTD
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

This technology significantly improves the sequencing depth of the CpG region and reduces the cost of sequencing, but limited by the endonuclease site, it cannot completely cover the CpG region
[0006] Moreover, in the traditional methylation sequencing based on bisulfite conversion, most of the cytosine (C) in the genome will be converted into thymine (T) after conversion, resulting in base imbalance. Greatly reduce the alignment rate of reads containing unmethylated cytosine, resulting in the inability to obtain a complete genome-wide methylation map, which limits practical applications

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  • Genome methylation library, preparation method and application thereof
  • Genome methylation library, preparation method and application thereof
  • Genome methylation library, preparation method and application thereof

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preparation example Construction

[0034] The method for preparing a genome methylation library according to an embodiment of the present invention includes the following steps S1-S5:

[0035] S1, providing a transposase complex (transposome), the transposase complex is composed of a transposable adapter and a transposase, and the transposable adapter contains a transposon sequence;

[0036] S2. Using a transposase complex to fragment genomic DNA (tagmentation) to obtain a fragmented product with a transposable adapter and a gap;

[0037] S3, using dNTPs that replace dCTP with mdCTP to perform gap filling (gap filling) on ​​the fragmented product;

[0038] S4, converting the fragmented product after the gap filling, so that the unmethylated cytosine is converted into uracil;

[0039] S5. Perform PCR amplification (amplification) on the transformed fragmented product to obtain a genome methylation library.

[0040] The present invention fragments the genomic DNA through the transposase complex and adds adapter...

Embodiment 1

[0058] Example 1 Genome methylation library construction mediated by Tn5 transposase

[0059] (1) Sample preparation: HEK293 cells were taken, and DNA was extracted using a cell genome DNA extraction kit. The resulting nucleic acids were dissolved in 50 μL TE buffer, and the concentration was measured using a Qubit 3.0 fluorometer. Finally, 50 ng DNA was taken for library construction according to the following operation steps.

[0060] (2) Tn5 processing mark:

[0061] a. Synthesize a nucleic acid fragment containing a 19bp transposase recognition sequence (the detailed sequence is shown in the following SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3), wherein part of the cytosine in sequence B and sequence C is formazan Modified cytosine ( C ). In sequence B and sequence C, "N" represents any base A, T, C or G, wherein cytosine is methylated modified cytosine.

[0062] Linker sequence A: CTGTCTCTTATACACATCT (SEQ ID NO: 1);

[0063] Linker sequence B: T C GT C GG C AG ...

Embodiment 2

[0084] Example 2 Construction of genome methylation library mediated by Tn5 transposase and nick-digested

[0085] (1) Sample preparation: HEK293 cells were taken, and DNA was extracted using a cell genome DNA extraction kit. The resulting nucleic acids were dissolved in 50 μL TE buffer, and the concentration was measured using a Qubit 3.0 fluorometer. Finally, 50 ng DNA was taken for library construction according to the following operation steps.

[0086] (2) Tn5 processing mark:

[0087]a. Synthesize a nucleic acid fragment containing a 19bp transposase recognition sequence (the detailed sequence is shown in the following SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3), wherein part of the cytosine in sequence B and sequence C is formazan Modified cytosine ( C ). In sequence B and sequence C, "N" represents any base A, T, C or G, wherein cytosine is methylated modified cytosine.

[0088] Linker sequence A: CTGTCTCTTATACACATCT (SEQ ID NO: 1);

[0089] Linker sequence B: T...

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Abstract

The invention relates to a genome methylation library, a preparation method and application thereof. The preparation method comprises the following steps: providing a transposase complex, wherein the transposase complex is composed of a transposase linker and a transposase, and the transposase linker contains a transposon sequence; carrying out fragmentation on genome DNA by using the transposase complex to obtain a fragmented product added with the transposase linker; carrying out notch completion on the fragmented product by using dNTPs in which dCTP is replaced by mdCTP; carrying out transformation treatment on the fragmented product after the notch completion, so that cytosine which is not subjected to methylation modification is transformed into uracil; and carrying out PCR amplification on the transformed fragmented product to obtain the genome methylation library. According to the invention, the problem of methylation library comparison analysis can be solved, more accurate methylation site analysis is realized, gene mutation site analysis can be carried out, library base balance can be greatly improved, and sequencing quality is greatly improved.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a genome methylation library and its preparation method and application. Background technique [0002] DNA methylation is a form of chemical modification of DNA that can alter genetic expression without altering the DNA sequence. The so-called DNA methylation refers to the covalent bonding of a methyl group at the 5th carbon position of cytosine of a genomic CpG dinucleotide under the action of DNA methyltransferase. A large number of studies have shown that DNA methylation can cause changes in chromatin structure, DNA conformation, DNA stability, and the interaction between DNA and proteins, thereby controlling gene expression. Abnormal methylation is a common epigenetic change in cancer. A large number of studies have shown that abnormal methylation is closely related to the occurrence, development, and carcinogenesis of tumors. [0003] The current methylation researc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B40/06C40B50/06C12Q1/6806C12Q1/6869
CPCC40B40/06C40B50/06C12Q1/6806C12Q1/6869
Inventor 阳卫超
Owner GUANGZHOU DRIPNANO BIOTECH CO LTD
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