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Product Brief

DNA methylation ( DNA methylation is one of the means of gene regulation. It plays a vital role in maintaining normal cell function, transmitting genomic imprinting, embryonic development, and tumorigenesis, and is also a hot topic in epigenetics research. Genome wide methylation sequencing ( Whole Genome Bisulfite Sequencing , WGBS) as a "gold standard" for methylation sequencing, a comprehensive, efficient and highly accurate methylation study of genomes with reference genomes is carried out at the whole genome level, thus constructing a single base resolution genome wide DNA methylation level map.

Mammalian DNA methylation is mainly CG type, and CpG Island rich in CpG dinucleotide is located near the transcriptional regulatory region, which is related to 56% of the human genome coding genes. 菜鸟竞技平台 ice and Agilent company launched the CpG Island precision methylation sequencing, using SureSelect system to enrich targets, covering the most epigenetic research areas in the genome, greatly improving the validity of sequencing data, achieving single base resolution, reducing costs, shortening the sequencing time, and achieving multiple sample experiments.


A workflow for Bisulfite Sequencing

Our advantages

1. precision coverage: the SureSelect target enrichment system can cover 3 million 700 thousand CpG, including CpG Island, GENCODE promoter, cancer and tissue-specific DMR, etc., to achieve a comprehensive coverage of methylation sites with biological significance.

Two High depth: just needed. The 10Gb data can reach 100 * coverage depth, 3 times the depth of ordinary sequencing, which greatly improves the detection rate of low frequency methylation sites.

Three High sensitivity: Adoption Bisulfite capture before transformation, methylation and non methylation equal capture; single base resolution, can accurately detect the methylation status of each cytosine.

4. low cost and high efficiency: the use of liquid capture technology can greatly improve the capture efficiency; at the same time, the selection strategy of target area can reduce the cost and achieve multi sample rapid experiment.

Sample requirements

Tissue samples More than 50mg

DNA sample

One Please provide the total amount. A sample of more than 3 g/, with a concentration of more than 100ng/ L DNA;

2. OD260/280 is between 1.8-2.0.

Three Electrophoretic detection was not obvious. The genome of RNA is clear and complete without degradation.

Four Please mark the sample number clearly when sending the sample. Parafilm membrane seal;

5. avoid repeated freezing and thawing during preservation.

6. dry ice transportation for sample delivery.

Note: there are differences between different samples. Please refer to the ice cream for more details.

Experiment flow


1. DNA extraction and quality control: gel electrophoresis quality control > Nanodrop quality control > Agilent 2200 quality control;

2. DNA ultrasound fragmentation: after fragmentation, end repair plus A, and connect methylation splice.

3. SureSelect target capture: single strand RNA probe hybridization for liquid phase capture.

4. Bisulfite Transformation: after targeted capture, BS transformation was performed to reduce methylation preference and DNA damage.

Five Joint amplification: after capture. PCR amplification library was constructed to reduce PCR preference.

6. on board sequencing: Strong ice recommended choice NovaSeq, dual terminal sequencing, has high flux, high base precision, low cost and fast speed. Data volume: 10G.

Data analysis process

Result example

1. Quality control of raw data
Aiming at the short sequence, pollution sequence, tail end low quality sequence, Raw Data is filtered and Fastp is used to control the quality of the data before and after filtration. The results of the analysis of base quality, GC content and length distribution are obtained. Figure 1 shows the quality control results of Raw Data.

Base quality results map

Note: the abscissa of the left picture represents the base site, and the ordinate represents the base mass value. The different color curves represent the mass values of different bases on each read; the right coordinates represent the base sites, the ordinates represent the ratio of base content, and the different color curves represent the bases of different sites.



2. Sequence alignment (DNA Mapping)
Bismark software was used to compare the filtered data chain to the reference genome. The methylation types, status and proportion of 5mC were obtained for subsequent methylation level, distribution and differential methylation analysis. The Samtools tool is used to sort and index the genome comparison files.

Comparison process

Note: the diagram is Bismark comparison schematic diagram.


3, methylation level and analysis
The methylation pattern, level and distribution of 5mC identification and analysis results were analyzed by Bismark algorithm, and the methylation types, levels and genomic distribution in different samples were obtained.

Genomic distribution of CpG locus

Li J et al., Genome Research. 2019

Note: the graph is compared. GPS and Genomic distribution of CpG loci detected by WGBS



4. Sample correlation, clustering and PCA analysis.
The results of 5mC identification analysis of each sample were taken as the research objects. The PCA methylation reduction algorithm, Pearson correlation coefficient and K-Mean clustering algorithm were used to describe the methylation degree and sample correlation of each methylation sequencing sample, and the correlation between the samples was obtained.

Correlation analysis of multiple methylation

Wan et al., Sci Rep. 2016

Note: this map shows the sexual and sexual relationship between skeletal muscle of tilapia. CpG Methylation Pearson correlation


5, differential methylation area (DMR) screening
Taking the 5mC identification analysis results of each sample as the research object, the Methykit algorithm was used to analyze the differential methylation region, and the methylation regions of different samples were obtained.

CIRCOS: differential methylation region

Yizan Ma et al., Plant Cell. 2018

Note: the distribution of DMR on different cotton lines (84021 and H05) at different temperatures (NT and HT) at different temperatures was shown by drawing the visual Circos map.



6. DMS/DMR related gene analysis
According to DMR annotation, DMR related genes were obtained, and then the GO, KEGG Pathway and PPI of related gene groups were analyzed according to the gene group.

PPI analysis of DMR related gene cluster

Note: Based on DMS/DMR Annotated gene GO entries and Pathway entries, we can draw GO-Tree and Pathway-Act-Network according to their upper and lower regulatory relationship. And based on PPI analysis of STRING database



Advanced data analysis
1, differential methylation area (DMR) notes
The ChipSeeker algorithm was used to annotate the regions of differential methylation and obtain DMR corresponding genes and their genetic structure, including promoters, exons, Chi Ko and intergenic regions.

Different samples Genomic distribution of CG-DMR

Rizzardi LF et al., Nat Neurosci. 2019

Note: the graph shows differences through cluster analysis. Cell type and Neuronal Medium Distribution of CG-DMR in genome structure



Examples of literature

[1] Ai T, et al. DNA methylation profile is associated with associated 2018. T 12


[2] Liu H, et al. Integrative analysis of DNA methylation, mRNAs, methylation, H, 17 (1): 105., 105.


[3] Fang X, et al. Comparative genome-wide methylation analysis of longissimus longissimus, X 2017 (3); 12 (8):


[4] Wang Z, et al. Decreased Methylation Level of H3K27me3 Increases H3K27me3 2017 Z; 54 (9):


[5] Cheng J, et al. SUMOylation of MeCP2 is essential for essential 2014 J; 128 (6):

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