Enzymatic Methyl Sequencing (EM-Seq)

Overview

Introduction to Enzymatic Methyl Sequencing (EM-Seq)

DNA methylation at the C5 position of cytosine plays a crucial role in gene expression and chromatin remodelling. As one of the most important research areas in epigenetics, methyl-seq tech is .With the development of high-throughput sequencing technology, WGBS has gradually become the gold standard for DNA methylation sequencing.

Enzymatic Methyl Sequencing (EM-seq) has been developed to overcome the shortcomings of WGBS, an enzyme-based method for the detection of 5mC and 5hmC at the single base level. The enzymatic conversion method is gentler and causes less DNA damage, thereby avoiding DNA molecule cleavage and loss.

EM-Seq Highlights

Enzyme based method Minimizes damage to DNA molecules
Less DNA input required
Less sequencing depth required
Superior performance compared to bisulfide sequencing
cfDNA is acceptable

EM-seq Specifications: DNA Sample Requirements

Sample Type	Amount	Purity or fragment size
Genomic DNA	≥ 50 ng	Fragments are above 5000 bp, and mainly above 13000 bp, no degradation, no contamination, no EDTA
cfDNA	≥ 50 ng	Agilent 2100 peak at 170bp and integer multiples, no genomic contamination, no contamination, no EDTA

EM-Seq Specifications: Sequencing and Analysis

Platform Type	Illumina NovaSeq platform
Read Length	Paired-end 150 bp
Sequencing Depth	≥ 20× coverage for the species with reference genome
Standard Data Analysis	Data Quality Control mCs detection, methylation level calculation Methylation level and frequency distribution Differentially Methylated Site (DMS) detection Differentially Methylated Regions (DMRs), Differentially Methylated Promoter (DMPs) detection and annotation Function enrichment of DMR-associated genes and DMP-associated genes Visualization of BS seq data Comparative analysis (among samples)
Co-Analysis with mRNA-seq (Integrated Analysis of Methylome and Transcriptome)	The distribution of gene methylation level and expression level at the chromosome level, and the distribution of methylation level and expression level at the upstream and downstream levels of genes. The relationship between DMR related gene’s expression and methylation modification The intersection of DMR related genes and differentially expressed genes, and display the methylation level, expression amount and annotation information of the intersection genes in different forms. Functional analysis of DMR related genes and differentially expressed genes, including GO, KEGG, and Motif Analysis.

Project Workflow of Novogene EM-Seq Service

The Novogene EM-Seq service comprises four steps: Sample preparation, library preparation, sequencing, and bioinformatics analysis, DNA extraction service is also available. The EM-seq conversion reaction is a two-step process: 1) TET2 and T4-BGT Protection of 5mC and 5hmC. 2) APOBEC then deaminates cytosines into uracils. Please contact us for more information about your EM-Seq projects.

To guarantee the accuracy and reliability of sequencing data, Novogene audits every experimental step through quality control, fundamentally ensuring high-quality data output from sampling to the final data report. This commitment to high-quality data is essential for the correctness, comprehensiveness, and credibility of bioinformatics analysis.

A genomic and epigenomic atlas of prostate cancer in Asian populations

NatureIssue Date: 25 March 2020IF: 42.778DOI: 10.1038/s41586-020-2135-x
- Li, J., Xu, C., Lee, H. J., Ren, S., Zi, X., Zhang, Z., … & Hou, J. (2020). A genomic and epigenomic atlas of prostate cancer in Asian populations. Nature, 580(7801), 93-99.
Single-Base Methylome Analysis Reveals Dynamic Epigenomic Differences Associated with Water Deficit in Apple

Plant Biotechnology JournalIssue Date: 10 August 2017IF: 8.154DOI: 10.1111/pbi.12820
- Xu, J., Zhou, S., Gong, X., Song, Y., van Nocker, S., Ma, F., & Guan, Q. (2018). Single‐base methylome analysis reveals dynamic epigenomic differences associated with water deficit in apple. Plant biotechnology journal, 16(2), 672-687.
Genome-wide Analysis of DNA Methylation Profiles in a Senescence-accelerated Mouse Prone 8 Brain using Whole-genome Bisulfite Sequencing

BioinformaticsIssue Date: 27 January 2017IF: 5.610DOI: 10.1093/bioinformatics/btx040
- Zhang, S., Qin, C., Cao, G., Guo, L., Feng, C., & Zhang, W. (2017). Genome-wide analysis of DNA methylation profiles in a senescence-accelerated mouse prone 8 brain using whole-genome bisulfite sequencing. Bioinformatics, 33(11), 1591-1595.
Altered Transcription and Neofunctionalization of Duplicated Genes Rescue the Harmful Effects of a Chimeric Gene in Brassica napus

The Plant CellIssue Date: 24 August 2016
- Xia, S., Wang, Z., Zhang, H., Hu, K., Zhang, Z., Qin, M., … & Shen, J. (2016). Altered transcription and neofunctionalization of duplicated genes rescue the harmful effects of a chimeric gene in Brassica napus. The Plant Cell, 28(9), 2060-2078.
Whole-genome analysis of 5-hydroxymethylcytosine and 5-methylcytosine at base resolution in the human brain

Genome BiologyIssue Date: 04 March 2014IF: 10.806DOI: 10.1186/gb-2014-15-3-r49
- Wen, L., Li, X., Yan, L., Tan, Y., Li, R., Zhao, Y., … & Yu, M. (2014). Whole-genome analysis of 5-hydroxymethylcytosine and 5-methylcytosine at base resolution in the human brain. Genome biology, 15(3), 1-17.

Enzymatic Methyl Sequencing (EM-Seq)

Introduction to Enzymatic Methyl Sequencing (EM-Seq)

EM-Seq Highlights

EM-seq Specifications: DNA Sample Requirements

EM-Seq Specifications: Sequencing and Analysis

Project Workflow of Novogene EM-Seq Service

Methylation Level Distribution on Whole Genome

Heatmap Analysis for Methylation Levels of Gene Functional Region

Methylation Level Distribution at Functional Genetic Elements

Circos Plot for DMR Condition in three contexts (CG, CHG, CHH)

Diagram of GO Enrichment

More Research Services

Reduced Representation Bisulfite Sequencing (RRBS)

ATAC-seq

RNA Immunoprecipitation Sequencing (RIP-seq)

Chromatin Immunoprecipitation Sequencing (ChIP-seq)

Whole Genome Bisulfite Sequencing (WGBS)