Screening of differential methylation region (DMR)
DMS/DMR related gene analysis
Annotation of differential methylation region (DMR)
Examples of literature
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 GenomeBisulfiteSequencing,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 andAgilent 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
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.
TwoHigh 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.
ThreeHigh sensitivity: AdoptionBisulfite 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.
Tissue samplesMore than 50mg
OnePlease 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.
ThreeElectrophoretic detection was not obvious.The genome of RNA is clear and complete without degradation.
FourPlease 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.
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.
FiveJoint amplification: after capture.PCR amplification library was constructed to reduce PCR preference.
6. on board sequencing:Strong ice recommended choiceNovaSeq, dual terminal sequencing, has high flux, high base precision, low cost and fast speed.Data volume: 10G.
Data analysis process
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.
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.GPSandGenomic 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.CpGMethylationPearson 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 onDMS/DMRAnnotated geneGO entries and Pathway entries, we can draw GO-Tree and Pathway-Act-Network according to their upper and lower regulatory relationship.And based onPPI 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 samplesGenomic distribution of CG-DMR
Rizzardi LF et al., Nat Neurosci. 2019
Note: the graph shows differences through cluster analysis.Cell typeandNeuronalMediumDistribution of CG-DMR in genome structure
Examples of literature
 Ai T, et al. DNA methylation profile is associated with associated 2018. T 12
 Liu H, et al. Integrative analysis of DNA methylation, mRNAs, methylation, H, 17 (1): 105., 105.
 Fang X, et al. Comparative genome-wide methylation analysis of longissimus longissimus, X 2017 (3); 12 (8):
 Wang Z, et al. Decreased Methylation Level of H3K27me3 Increases H3K27me3 2017 Z; 54 (9):
 Cheng J, et al. SUMOylation of MeCP2 is essential for essential 2014 J; 128 (6):