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Updated: Apr 6, 2026
DNA Methylation: Bisulphite Modification and Analysis
Published on: October 21, 2011
Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing
Miao Yu1, Lexiang Ji2, Drexel A Neumann3
1Department of Chemistry and Institute for Biophysical Dynamics, Howard Hughes Medical Institute, The University of Chicago, Chicago, IL 60637, USA.
Abstract:
Restriction-modification (R-M) systems pose a major barrier to DNA transformation and genetic engineering of bacterial species. Systematic identification of DNA methylation in R-M systems, including N(6)-methyladenine (6mA), 5-methylcytosine (5mC) and N(4)-methylcytosine (4mC), will enable strategies to make these species genetically tractable. Although single-molecule, real time (SMRT) sequencing technology is capable of detecting 4mC directly for any bacterial species regardless of whether an assembled genome exists or not, it is not as scalable to profiling hundreds to thousands of samples compared with the commonly used next-generation sequencing technologies. Here, we present 4mC-Tet-assisted bisulfite-sequencing (4mC-TAB-seq), a next-generation sequencing method that rapidly and cost efficiently reveals the genome-wide locations of 4mC for bacterial species with an available assembled reference genome. In 4mC-TAB-seq, both cytosines and 5mCs are read out as thymines, whereas only 4mCs are read out as cytosines, revealing their specific positions throughout the genome. We applied 4mC-TAB-seq to study the methylation of a member of the hyperthermophilc genus, Caldicellulosiruptor, in which 4mC-related restriction is a major barrier to DNA transformation from other species. In combination with MethylC-seq, both 4mC- and 5mC-containing motifs are identified which can assist in rapid and efficient genetic engineering of these bacteria in the future.
Insights
This study introduces 4mC-Tet-assisted bisulfite-sequencing (4mC-TAB-seq), a new method for genome-wide N(4)-methylcytosine (4mC) detection in bacteria. This advance aids genetic engineering by identifying methylation patterns that hinder DNA transformation.
Area of Science:
- Genomics
- Molecular Biology
- Microbial Genetics
Background:
- Restriction-modification (R-M) systems impede bacterial genetic engineering by restricting foreign DNA.
- Identifying DNA methylation patterns, including N(6)-methyladenine (6mA), 5-methylcytosine (5mC), and N(4)-methylcytosine (4mC), is crucial for overcoming these barriers.
- Existing methods like single-molecule, real time (SMRT) sequencing are not scalable for high-throughput analysis.
Purpose of the Study:
- To develop a scalable, cost-effective next-generation sequencing (NGS) method for genome-wide N(4)-methylcytosine (4mC) detection in bacteria.
- To enable systematic identification of DNA methylation in bacterial species, facilitating genetic engineering strategies.
- To characterize 4mC methylation in the hyperthermophilic genus Caldicellulosiruptor.
Main Methods:
- Development of 4mC-Tet-assisted bisulfite-sequencing (4mC-TAB-seq), an NGS method for bacterial DNA methylation profiling.
- 4mC-TAB-seq distinguishes 4mC from other cytosine modifications (cytosine and 5mC) by converting them to thymine, while 4mC is read as cytosine.
- Application of 4mC-TAB-seq and MethylC-seq to study methylation in Caldicellulosiruptor.
Main Results:
- 4mC-TAB-seq accurately and efficiently maps genome-wide 4mC locations in bacteria with available reference genomes.
- The method was successfully applied to Caldicellulosiruptor, a genus where 4mC poses a significant transformation barrier.
- Combined analysis with MethylC-seq identified both 4mC- and 5mC-containing motifs.
Conclusions:
- 4mC-TAB-seq provides a scalable and cost-effective NGS solution for bacterial 4mC profiling.
- The identified methylation motifs in Caldicellulosiruptor can guide future genetic engineering efforts.
- This approach enhances the tractability of bacterial species for genetic manipulation.
