Long-Read Sequencing of DNA Methylation

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Long-Read Sequencing of DNA Methylation

DNA methylation analysis can help researchers and professionals gain insight into gene regulation as well as identify potential biomarkers. DNA methylation tends to occur in a relatively wide range of genomic regions in both normal and disease conditions. However, the current understanding of DNA methylation is still pieced together from fragmented information obtained by short-read sequencing. Emerging third-generation sequencing technologies (PacBio SMRT and Nanopore sequencing) can directly detect DNA methylation modifications. Using these newer sequencing platforms, CD Genomics is dedicated to providing a comprehensive elucidation of DNA methylation, providing valuable information on gene expression regulation at the chromosomal level.

DNA Methylation Analysis Overview

DNA methylation plays an essential role in epigenetic regulation of cells, such as X-chromosome inactivation, genomic imprinting, transposon suppression, and tumorigenesis, and is one of the hot spots of current research. DNA methylation is a biological process in which methyl groups are covalently incorporated into DNA molecules. 5-Methylcytosine (5mC) is the most common form of this process and is the most studied and best understood modification in plants and animals. N6-methyladenine (6mA), 4-methylcytosine (4mC), and 5mC are frequent in bacteria, having an impact on physiology as well as virulence.

Currently, to detect 5mC, the combination of bisulfite treatment and massively parallel DNA sequencing, namely, bisulfite sequencing (BS-seq) is the most widely used. However, this method has a number of disadvantages, including lack of specificity (inability to distinguish between 5mC and 5-hydroxymethylcytosine, 5hmC), short reads with low sequence diversity, DNA degradation, and the need for specialized protocols. PacBio SMRT sequencing and Oxford Nanopore sequencing are two main third-generation sequencing methods. They allow unambiguous mapping of repetitive and complex regions of the genome and have been widely utilized to detect structural variants, perform phasing haplotypes, and assemble genomes.

Detection of base modifications by bisulfite and long-read sequencing. Fig1. Detection of base modifications by bisulfite and long-read sequencing. (Gouil, Q. and Keniry, A., 2019)

Long-Read Whole-Genome Analysis of DNA Methylation

SMRT and nanopore long-read sequencing enable the simultaneous detection of a range of base modifications without the need for additional sample preparation. Each method has its own detection focus.

  • PacBio SMRT sequencing is most sensitive for 4mC and 6mA detection and is suitable for bacterial epigenomics.
  • Oxford Nanopore sequencing is capable of detecting a range of base modifications including 6mA, 5mC, 5hmC, and BrdU, and performs best in detecting 5mC in particular. Compared to SMRT sequencing, nanopore sequencing has a lower cost per Gb and is suitable for larger genomes.

Sample Requirements

-Sample type: conventional plant and animal samples.
-DNA amount: ≥ 10 μg, sample concentration ≥ 100 ng/µl.
-Sequencing strategy: non-PCR amplification library construction, Nanopore PromethION and PacBio Sequel ll platform.

Workflow of Our Service

Workflow of Our Service-CD Genomics.

Analysis Contents

  • Data re-processing and QC.
  • Reference sequence alignment.
  • Detection of different types of base modification sites.
  • Base modification degree analysis.
  • Regional analysis of differential methylation.
  • Functional analysis of base modified genes.

DNA methylation, a common epigenetic modification, affects the regulation of gene expression and is involved in a variety of human diseases. DNA methylation is also a common topic in the field of agricultural genomics and can be used to explore responses to drought, extreme temperatures, and other environmental changes. If you are interested in our services, please don't hesitate to contact us. We've got everything covered for your needs and are ready to assist.


  1. Gouil, Q., & Keniry, A. (2019). "Latest techniques to study DNA methylation." Essays in biochemistry, 63(6), 639-648.
  2. Tse, O. O., et al. (2021). "Genome-wide detection of cytosine methylation by single molecule real-time sequencing." Proceedings of the National Academy of Sciences, 118(5).
For Research Use Only. Not for use in diagnostic procedures.

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