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Updated: Mar 15, 2026

Targeted DNA Methylation Analysis by Next-generation Sequencing

Targeted DNA Methylation Analysis by Next-generation Sequencing

Published on: February 24, 2015

tRNA base methylation identification and quantification via high-throughput sequencing.

Wesley C Clark¹, Molly E Evans¹, Dan Dominissini²

¹Department of Biochemistry and Molecular Biology.

RNA (New York, N.Y.)

|September 11, 2016

Summary

This summary is machine-generated.

This study introduces a new sequencing method to quantify tRNA base methylations. The approach identifies new methylation sites and their fractional levels in human cells, advancing tRNA modification research.

Keywords:

high-throughput sequencing methylation modification quantification tRNA

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Methodology for Accurate Detection of Mitochondrial DNA Methylation

Published on: May 20, 2018

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Last Updated: Mar 15, 2026

Targeted DNA Methylation Analysis by Next-generation Sequencing

Targeted DNA Methylation Analysis by Next-generation Sequencing

Published on: February 24, 2015

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

Published on: February 24, 2015

Methodology for Accurate Detection of Mitochondrial DNA Methylation

Methodology for Accurate Detection of Mitochondrial DNA Methylation

Published on: May 20, 2018

Area of Science:

Molecular Biology
Genomics
Biochemistry

Background:

Eukaryotic transfer RNAs (tRNAs) have numerous modifications crucial for their function.
Base methylation is a key tRNA modification, but quantitative analysis of its fraction has been limited.
Existing methods for identifying tRNA methylations are often qualitative and lack fractional information.

Purpose of the Study:

To develop and validate a high-throughput sequencing method for identifying and quantifying tRNA base methylations.
To determine the modification fraction of tRNA bases at single-base resolution in human cells.
To discover novel tRNA methylation sites and analyze their dynamics.

Main Methods:

Utilized a recently developed high-throughput sequencing method (DM-tRNA-seq).
Employed a combination of demethylase treatment and cDNA synthesis with thermophilic reverse transcriptase.
Developed a quantitative "Modification Index" (MI) integrating base mutation and positional stop data from sequencing.

Main Results:

Identified and quantified six base methylations in human tRNA and rRNA with single-base resolution.
Discovered numerous novel methylation sites in both nuclear and mitochondrial-encoded tRNAs.
Validated the quantitative nature of the Modification Index (MI) using primer extension assays.

Conclusions:

The DM-tRNA-seq method provides a quantitative approach to study tRNA base methylations.
This method enables the systematic analysis of tRNA modification fractions and dynamics.
The approach is broadly applicable for tRNA methylation site identification and comparative analyses across different samples.