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Updated: Aug 27, 2025
13:47
Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution
Published on: February 24, 2015
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Post-bisulfite Adaptor Tagging Based on an ssDNA Ligation Technique (tPBAT)
Fumihito Miura1, Yukiko Shibata2, Miki Miura2
1Department of Biochemistry, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan. fumihito@med.kyushu-u.ac.jp.
Methods in Molecular Biology (Clifton, N.J.)
|September 29, 2022
Summary
We developed tPBAT, a new DNA sequencing protocol that improves data quality and efficiency for methylome analysis from small samples. This method overcomes limitations of previous protocols, offering longer DNA inserts and higher mapping rates.
Area of Science:
- Epigenetics
- Molecular Biology
- Genomics
Background:
- Post-bisulfite adaptor tagging (PBAT) is crucial for preparing sequencing libraries from bisulfite-treated DNA.
- Previous PBAT and rPBAT protocols, while sensitive for single-cell methylome analysis, suffered from drawbacks due to repeated random priming.
Purpose of the Study:
- To introduce a novel protocol, tPBAT, designed to overcome the limitations of existing PBAT methods.
- To detail the application of tPBAT for analyzing small DNA samples, particularly in methylome studies.
Main Methods:
- Development of a unique single-strand DNA ligation technique, termed TACS ligation.
- Establishment and implementation of the tPBAT protocol utilizing TACS ligation.
- Application of tPBAT to small sample volumes for methylome analysis.
Main Results:
- tPBAT demonstrated improved data quality compared to rPBAT.
- The protocol yielded longer DNA inserts and a higher mapping rate.
- tPBAT natively supports paired-end sequencing and indexing.
Conclusions:
- tPBAT represents a significant advancement in DNA library preparation for methylome analysis.
- The protocol offers enhanced efficiency and data quality, especially for limited sample quantities.
- tPBAT is a valuable tool for epigenomic research requiring sensitive and accurate methylome profiling.
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