ONT-Based RNA Methylation Sequencing

Third-generation Sequencing

Service Overview

Transcriptome-wide RNA modification mapping has revolutionized our knowledge in their profoundly impact on biological processes. RNA modifications, such as N6-adenylate methylation (m6A), n1-adenylate methylation (m1A), Cytosine hydroxylation (m5C),etc., have attracting growing attention on their roles in regulatory level. RNA m6A, which refers to methylation on the 6th nitrogen of adenylate, is one of the most abundant RNA modifications, which account for approximately 80% of total RNA modifications. When RNA molecules with different decoration on nucleotides pass through a nano-sized protein pore (Nanopore), distinct ionic currents can be generated, which enables us to record and translate these signals into specific nucleotides and corresponding modifications. ONT-based RNA methylation sequencing is a method empowers us to read transcriptome-wide RNA modifications at single-nucleotide resolution, which is free from antibody and any synthesis steps.
Experimental workflow

Bioinformatic analysis

Service Applications

Gene structure study
Including alternative splicing, APA, Fusion genes, SSR, CDS prediction, TSS/TES identification, etc.

Gene Function study
Identifying biological function related modifications in particular tissues.

Full-length transcripts quantification
Quantifying full-length transcripts to reveal differentially expressed transcripts; Identifying relevant functional genes.

Specific expression of allele genes
Identifying unequal allelic expression. Samples with same genome but distinct phenotypes may due to unequal allelic expression. It is also a key marker in cancer.

RNA methylation
Amplication-free full-length transcriptome sequencing and reading of transcriptome-wide RNA modifications, such as m6A.

Results Demo

Violin plot on transcript methylation level

The violin plot shows the overall distribution of transcripts methylation level in each samples. X-axis: Sample; Y-axis: transcripts methylation level; Each colour represents a sample.

Correlation on methylation level

Correlation heatmap reveals the similarities (correlations) on methylation level of each site among samples.

Differential methlation loci

Differential methylated loci (DML) can be identified between samples and summarized into hyper-methylated DMLs and hypo-methylated DMLs.

DML Hierarchical clustering

Hierarchical clustering will be processed on DMLs identified.


1What is methylation level?
In this service, transcript methylation level is defined as average methylation level of all sites on the transcript.
2Which molecules are involved in RNA modification?
a. Writer: molecules induced methylation. METTL3 and METTL4 are two of the most commonly studied molecules that can catalyze methylation on mRNA N6-andenylate both in vivo and in vitro. WTAP is another key component in methyl-transferase complex.

b. Erasers: molecules removed methylation. FTO and ALKBH5 have been reported to remove m6A from mRNA.

c. Readers: molecules recognize methylation and participate in downstream RNA processes, such as translation and degradation. YTHDF domain is able to recognize and bind to m6A, which facilitate RNA degradation.