ONT-based DNA Methylation Sequencing

Nanopore-Based DNA methylation Sequencing

Service Overview

Nanopore sequencing distinguishes itself from other sequencing platforms, in that the nucleotides are read directly without DNA synthesis process. As a single strand DNA passes through a nano-sized protein pore (nanopore), different nucleotides generate different ionic current, which can be captured and transferred into sequence of bases. Synthesis-free process of sequence reading largely preserved DNA methylation information on template. Methylated ATCG generate distinct ionic currents from un-methylated ones, which can be read directly by the platform. Therefore, Nanopore sequencing empowers whole-genome profiling of both 5mC and 6mA at single-nucleotide resolution. Moreover, the dataset generated here can also be processed for re-sequencing and reveal structural variations simultaneously.

Project Workflow

Sample delivery
Library construction and sequencing
Data analysis
Data analysis
After-sale technical support






Experimental Workflow

Sample quality control, library construction, library quality control and sequencing are all processed following protocol provided by Oxford Nanopore Technologies (ONT).

Bioinformatic Analysis


Results Demo

Methylation level of CpG island

CpG island (CGI) is defined as regions rich in CpG. Average methylation level is calculated for CGI regions and their upper and down stream.
Motif sequence of 6mA regions

Motif information is collected at 6mA site within regions with high 6mA methylation level. The height of the base represents relative frequency of specific nucleotide on the site.









Whole genome methylation distribution at chromosome-level

Average methylation level is calculated for each frame of chromosomes, The figure presented the genome-wide distribution of methylation.









Correlation analysis between samples

Pearson correlation coefficient of methylation level is calculated for each two samples to examine the similarity of the samples in methylation level.






GO enrichment analysis on DMR related genes DMR related genes are defined as genes with DMR annotated on the gene but not in intergenic regions. Potent DNA methylation related biological processes can be identified by enrichment analysis on DMR related genes.

FAQ

1I only have limited amount of sample. Can I use PCR products for methylation sequencing?
Answer: No. PCR will eliminate DNA methylation information from original DNA extracts. ONT reads different ionic current generated by different bases and methylated bases. Therefore, PCR products can not be used as samples in this service.
2What is 6mA?
Answer: 6mA stands for adenines, on which N6 is methylated (N6-methyladenine). 5mC has been extensively studied in eukaryotic species with help of NGS. Taking advantage of ONT-sequencing platform, 6mA profiling can be achieved. Evidence is accumulated to show that 6mA plays pivotal role in biological processes related to development and disease. There are more to be explored.
3What's the difference between Nanopore DNA methylation sequencing and 6mA-IP-SEQ?
Answer: In 6mA-IP-SEQ, anti-6mA antibody is applied to pooling down the fragments with 6mA and process sequecing on the extracts. However, it can only identify the regions with 6mA but not at single-nucleotide resolution. Nanopore sequencing identifies methylated bases according to the distinct ionic current generated by different bases and quantifies the methylation level by counting the reads supporting the methylation. It empowers 6mA profiling at single-nucleotide resolution and discovering DMR between samples, which contributes to the research on DNA-methylation related functions.