阅读说明：本技术 一种用于评估幼年型粒单核细胞白血病预后的全基因组dna甲基化检测方法 (Whole genome DNA methylation detection method for evaluating prognosis of juvenile myelomonocytic leukemia ) 是由 杨文钰 张英驰 竺晓凡 于 2020-04-29 设计创作，主要内容包括：本发明公开了一种用于评估幼年型粒单核细胞白血病预后的全基因组DNA甲基化检测方法,包括以下步骤：a.文库构建；b.生物信息分析；c.构建全基因组DNA甲基化图谱。本发明通过检测DNA甲基化水平预测JMML预后,JMML患者异常高甲基化水平提示预后不良,本发明建立JMML DNA甲基化检测方法对于JMML预后评估具有重要的意义,去甲基化药物也有望成为高甲基化水平JMML患者治疗的重要药物之一。本发明为JMML预后评估及药物疗效预测奠定了理论基础和试验基础,具有广阔的应用前景。(The invention discloses a whole genome DNA methylation detection method for evaluating prognosis of juvenile myelomonocytic leukemia, which comprises the following steps of a, library construction, b, biological information analysis and c, whole genome DNA methylation map construction, the method predicts JMM L prognosis by detecting DNA methylation level, and prompts poor prognosis by abnormal hypermethylation level of JMM L patients, the JMM L DNA methylation detection method established by the invention has important significance for JMM L prognosis evaluation, demethylation drugs are expected to become one of important drugs for treating the JMM L patients with the hypermethylation level, theoretical basis and experimental basis are laid for JMM L prognosis evaluation and drug efficacy prediction, and the method has wide application prospect.)

1. A whole genome DNA methylation assay for assessing the prognosis of juvenile myelomonocytic leukemia, characterized by: the method comprises the following steps:

a. library construction

DNA extraction: extracting nucleic acid from the sample DNA by using a genome DNA extraction kit;

ii.construction of genomic libraries

I) sample disruption: the sample disruption method is a Covaris disruption method, and can break the sample DNA into fragments in the range of 100-300 bp;

ii) repairing the tail end of the DNA fragment, adding A basic group at the 3' end, and connecting a sequencing adaptor;

iii) performing Bisulite treatment by adopting ZYMO EZ DNA Methylation-Gold kit;

iv) cutting and recovering the gel after treatment, and selecting the size of the library fragment;

v) using the qualified library for on-machine sequencing;

b. biological information analysis

Analyzing standard information;

ii, data statistics and quality control;

methylation level analysis of C base;

iv, the distribution trend of the whole genome methylation level;

i) distribution ratio among methylated C bases;

ii) methylation level of all C;

iii) methylation level of C in each chromosome;

iv) counting the methylation level of C in CG, CHG and CHH in different gene regions;

v) methylation levels of C in CG, CHG and CHH in different gene element regions;

vi) sequence feature analysis near methylated C in CHG and CHH;

c. construction of Whole genome DNA methylation map

The density distribution of methylated C bases at the chromosome level;

methylated C base density distribution of Scaffold;

methylation distribution characteristics of different genomic regions;

iv analysis of regions of differential methylation of DNA methylation levels in different transcription elements of the genome.

2. The method of claim 1 for detecting genome-wide DNA methylation for assessing the prognosis of juvenile myelomonocytic leukemia, wherein the method comprises the steps of: after the library was constructed, a filter of data was performed.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a whole genome DNA methylation detection method for evaluating prognosis of juvenile myelomonocytic leukemia.

Background

The prognosis is definite, the prognosis index of the disease is limited at present, the JMM L mutant gene has certain indication significance for prognosis, but the prognosis difference of the same gene is obvious, for example, partial disease of NRAS gene mutation patients is rapid in progress and fierce, only the possibility of curing the disease by Hematopoietic Stem Cell Transplantation (HSCT) exists, partial patients are stable in disease, and spontaneous remission can be obtained.

Recent studies have shown that epigenetics plays a major role in the pathogenesis and prognosis evaluation of tumors. DNA methylation is a widely studied epigenetic modification that, together with histone modifications, plays an important role in regulating gene expression and chromatin conformation. Generally, methylated DNA refers to 5-methylcytosine (5mC), which is a structure formed by adding a methyl group to the 5' C position of cytosine by the action of DNA methyltransferase (DNMT) (Vertino PM, 1996). Methylation in mammalian cells occurs mainly on the cytosine of the CG dinucleotide (Goldberg AD, 2007), while a large proportion of non-CG (CHH, CHG, H stands for A, C, T) methylation is present in plant cells (Jackson JP, 2002). In general, high DNA methylation levels inhibit gene expression, and demethylation allows for gene re-expression. DNA methylation is involved in numerous cell life activities including cell differentiation, tissue-specific gene expression, genomic imprinting, X chromosome inactivation, etc. (Bird A, 2002; Jones PA, 2001; Reikw, 2003). Abnormal DNA methylation can lead to the development of dysplasia, tumors, and other diseases. Therefore, the research on DNA methylation is of great significance for deeply understanding gene expression, ontogeny and the occurrence and development mechanisms of diseases. The research finds that the methylation of cytosine in CpG dinucleotide of promoter is an important negative regulatory factor for the gene expression of human genome. Aberrant methylation of tumor suppressor genes plays a crucial role in the pathogenesis of malignant tumors, including myeloid tumors and myelodysplastic syndromes.

Disclosure of Invention

The present invention provides a whole genome DNA methylation detection method for evaluating the prognosis of juvenile myelomonocytic leukemia, which aims to solve the technical problems.

The invention is realized by the following technical scheme:

a whole genome DNA methylation assay for assessing the prognosis of juvenile myelomonocytic leukemia comprising the steps of:

a. library construction

DNA extraction: extracting nucleic acid from the sample DNA by using a genome DNA extraction kit;

ii.construction of genomic libraries

I) sample disruption: the sample disruption method is a Covaris disruption method, and can break the sample DNA into fragments in the range of 100-300 bp;

ii) repairing the tail end of the DNA fragment, adding A basic group at the 3' end, and connecting a sequencing adaptor;

iii) performing Bisulite treatment by adopting ZYMO EZ DNA Methylation-Gold kit;

iv) cutting and recovering the gel after treatment, and selecting the size of the library fragment;

v) using the qualified library for on-machine sequencing;

b. biological information analysis

Analyzing standard information;

ii, data statistics and quality control;

methylation level analysis of C base;

iv, the distribution trend of the whole genome methylation level;

i) distribution ratio among methylated C bases;

ii) methylation level of all C;

iii) methylation level of C in each chromosome;

iv) counting the methylation level of C in CG, CHG and CHH in different gene regions;

v) methylation levels of C in CG, CHG and CHH in different gene element regions;

vi) sequence feature analysis near methylated C in CHG and CHH;

c. construction of Whole genome DNA methylation map

The density distribution of methylated C bases at the chromosome level;

methylated C base density distribution of Scaffold;

methylation distribution characteristics of different genomic regions;

iv analysis of regions of differential methylation of DNA methylation levels in different transcription elements of the genome.

Further, after the library was constructed, data was filtered.

The invention has the beneficial effects that:

the invention discloses a detection method for clinically evaluating prognosis of juvenile myelomonocytic leukemia, which predicts JMM L prognosis by detecting DNA methylation level, provides theoretical basis and experimental basis for JMM L prognosis evaluation and treatment of methylation drugs, and has wide application prospect in JMM L prognosis and treatment research.

Drawings

FIG. 1 is a flow chart of information analysis of the present invention.

Detailed Description

In order to make the technical solutions of the present invention better understood by those skilled in the art, the technical solutions of the present invention are further described below by way of specific embodiments with reference to the drawings of the specification.

Firstly, a library construction strategy: a small fragment library.

1.1 sequencing strategy: the sequencing platform was hiseq xten

1.2 Experimental flow:

DNA extraction and quality control

1) DNA extraction

And (3) extracting nucleic acid from the sample DNA by using the genome DNA extraction kit, and if the sample is the extracted DNA, skipping the step.

Genomic library construction

1) Sample disruption

The current sample disruption method is a Covaris disruption method, and can break sample DNA into fragments in the range of 100-300 bp. If the sample delivered is DNA that has been broken to the appropriate size, this step can be skipped.

2) Repairing the tail end of the DNA fragment, adding A base at the 3' end, and connecting a sequencing adaptor;

3) performing Bisulite treatment by adopting ZYMO EZ DNA Methylation-Gold kit;

4) after treatment, cutting and recovering gel, and selecting the size of the library fragment;

5) and (4) using the qualified library for on-machine sequencing, and if the sample grade is an unqualified sample, not performing the next experiment.

Second, information analysis process

2.1 BS analysis strategy: comparison of

2.2 information analysis flow chart (FIG. 1)

2.3 data Filtering

And (3) filtering sequencing data: removing reads containing the adapter; removing reads with the N proportion of more than 10 percent;

removing low-quality reads (the number of bases with quality value Q less than or equal to 5 accounts for more than 50% of the whole read); the duplification is removed.

Data requirements are as follows: q20% > 85%.

Third, biological information analysis content

3.1 Standard information analysis

1. Data statistics and quality control

2. Methylation level analysis of C base

3. Genome-wide methylation horizontal distribution trend

3.1 distribution ratio among methylated C bases

3.2 methylation levels of all C

3.3 methylation level of C in each chromosome (this analysis is only for species with complete chromosomal information)

3.4 statistics of C methylation levels in CG, CHG and CHH in different Gene regions

3.5 methylation levels of C in CG, CHG and CHH in regions of different Gene elements

3.6 sequence characterization of CHG, around methylated C in CHH

Four, whole genome DNA methylation map

4.1 Density distribution of methylated C bases at the chromosomal level (this analysis is only for species with complete chromosomal information)

4.2 methylated C base Density distribution of Scaffold (this analysis is for species with incomplete chromosomal information)

4.3 methylation profiles of different genomic regions

4.4 Differential Methylation Region (DMR) analysis of DNA methylation levels in different transcription elements of the genome.

The applicant declares that the above description is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be understood by those skilled in the art that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are within the scope and disclosure of the present invention.