Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite-sequencing
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 N6-methyladenine (6mA), 5-methylcytosine (5mC) and N4-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. Lastly, 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.
- Authors:
-
- The Univ. of Chicago, Chicago, IL (United States)
- The Univ. of Georgia, Athens, GA (United States)
- The Univ. of Georgia, Athens, GA (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
- Publication Date:
- Research Org.:
- Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER)
- OSTI Identifier:
- 1256683
- Grant/Contract Number:
- R01 HG006827
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Nucleic Acids Research
- Additional Journal Information:
- Journal Volume: 43; Journal Issue: 21; Journal ID: ISSN 0305-1048
- Publisher:
- Oxford University Press
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES
Citation Formats
Yu, Miao, Ji, Lexiang, Neumann, Drexel A., Chung, Dae -Hwan, Groom, Joseph, Westpheling, Janet, He, Chuan, and Schmitz, Robert J. Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite-sequencing. United States: N. p., 2015.
Web. doi:10.1093/nar/gkv738.
Yu, Miao, Ji, Lexiang, Neumann, Drexel A., Chung, Dae -Hwan, Groom, Joseph, Westpheling, Janet, He, Chuan, & Schmitz, Robert J. Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite-sequencing. United States. https://doi.org/10.1093/nar/gkv738
Yu, Miao, Ji, Lexiang, Neumann, Drexel A., Chung, Dae -Hwan, Groom, Joseph, Westpheling, Janet, He, Chuan, and Schmitz, Robert J. Wed .
"Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite-sequencing". United States. https://doi.org/10.1093/nar/gkv738. https://www.osti.gov/servlets/purl/1256683.
@article{osti_1256683,
title = {Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite-sequencing},
author = {Yu, Miao and Ji, Lexiang and Neumann, Drexel A. and Chung, Dae -Hwan and Groom, Joseph and Westpheling, Janet and He, Chuan and Schmitz, Robert J.},
abstractNote = {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 N6-methyladenine (6mA), 5-methylcytosine (5mC) and N4-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. Lastly, 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.},
doi = {10.1093/nar/gkv738},
journal = {Nucleic Acids Research},
number = 21,
volume = 43,
place = {United States},
year = {Wed Jul 15 00:00:00 EDT 2015},
month = {Wed Jul 15 00:00:00 EDT 2015}
}
Free Publicly Available Full Text
Publisher's Version of Record
Other availability
Search WorldCat to find libraries that may hold this journal
Cited by: 53 works
Citation information provided by
Web of Science
Web of Science
Save to My Library
You must Sign In or Create an Account in order to save documents to your library.
Works referenced in this record:
Human DNA methylomes at base resolution show widespread epigenomic differences
journal, October 2009
- Lister, Ryan; Pelizzola, Mattia; Dowen, Robert H.
- Nature, Vol. 462, Issue 7271
Identification and characterization of CbeI, a novel thermostable restriction enzyme from Caldicellulosiruptor bescii DSM 6725 and a member of a new subfamily of HaeIII-like enzymes
journal, May 2011
- Chung, Dae-Hwan; Huddleston, Jennifer R.; Farkas, Joel
- Journal of Industrial Microbiology & Biotechnology, Vol. 38, Issue 11
MethylC-seq library preparation for base-resolution whole-genome bisulfite sequencing
journal, February 2015
- Urich, Mark A.; Nery, Joseph R.; Lister, Ryan
- Nature Protocols, Vol. 10, Issue 3
Genomics of DNA cytosine methylation in Escherichia coli reveals its role in stationary phase transcription
journal, January 2012
- Kahramanoglou, Christina; Prieto, Ana I.; Khedkar, Supriya
- Nature Communications, Vol. 3, Issue 1
Tet-Mediated Formation of 5-Carboxylcytosine and Its Excision by TDG in Mammalian DNA
journal, August 2011
- He, Y. -F.; Li, B. -Z.; Li, Z.
- Science, Vol. 333, Issue 6047
Highlights of the DNA cutters: a short history of the restriction enzymes
journal, October 2013
- Loenen, Wil A. M.; Dryden, David T. F.; Raleigh, Elisabeth A.
- Nucleic Acids Research, Vol. 42, Issue 1
Base-Resolution Analysis of 5-Hydroxymethylcytosine in the Mammalian Genome
journal, June 2012
- Yu, Miao; Hon, Gary C.; Szulwach, Keith E.
- Cell, Vol. 149, Issue 6
Zeroing in on DNA methylomes with no BS
journal, June 2010
- Ecker, Joseph R.
- Nature Methods, Vol. 7, Issue 6
Cleavage of methylated CCCGGG sequences containing either N4-methylcytosine or 5-methytcytosine with Mspl, Hpall, Smal, Xmal and Cfr9I restriction endonudeases
journal, January 1987
- Butkus, V.; Petrauskiene, L.; Maneliene, Z.
- Nucleic Acids Research, Vol. 15, Issue 17
Cytosine modification in DNA by Bcn I methylase yields N 4 -methylcytosine
journal, September 1983
- Janulaitis, A.; Klimašauskas, S.; Petrušyte, M.
- FEBS Letters, Vol. 161, Issue 1
‘Leveling’ the playing field for analyses of single-base resolution DNA methylomes
journal, December 2012
- Schultz, Matthew D.; Schmitz, Robert J.; Ecker, Joseph R.
- Trends in Genetics, Vol. 28, Issue 12
Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning
journal, February 2008
- Cokus, Shawn J.; Feng, Suhua; Zhang, Xiaoyu
- Nature, Vol. 452, Issue 7184
N4-methylcytosine as a minor base in bacterial DNA.
journal, January 1987
- Ehrlich, M.; Wilson, G. G.; Kuo, K. C.
- Journal of Bacteriology, Vol. 169, Issue 3
DNA methylation in thermophilic bacteria: N 4 -methylcytosine, 5-methylcytosine, and N 5 methyladenine
journal, January 1985
- Ehrlich, Melanie; Gama-Sosa, Miguel A.; Carreira, Laura H.
- Nucleic Acids Research, Vol. 13, Issue 4
Mechanisms of, and Barriers to, Horizontal Gene Transfer between Bacteria
journal, September 2005
- Thomas, Christopher M.; Nielsen, Kaare M.
- Nature Reviews Microbiology, Vol. 3, Issue 9
Enhanced 5-methylcytosine detection in single-molecule, real-time sequencing via Tet1 oxidation
journal, January 2013
- Clark, Tyson A.; Lu, Xingyu; Luong, Khai
- BMC Biology, Vol. 11, Issue 1
Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine
journal, November 2012
- Yu, Miao; Hon, Gary C.; Szulwach, Keith E.
- Nature Protocols, Vol. 7, Issue 12
Bisulfite Sequencing Protocol Displays both 5-Methylcytosine and N4-Methylcytosine
journal, June 1999
- Vilkaitis, Giedrius; Klimašauskas, Saulius
- Analytical Biochemistry, Vol. 271, Issue 1
Epigenome-wide inheritance of cytosine methylation variants in a recombinant inbred population
journal, June 2013
- Schmitz, R. J.; He, Y.; Valdes-Lopez, O.
- Genome Research, Vol. 23, Issue 10
Tet Proteins Can Convert 5-Methylcytosine to 5-Formylcytosine and 5-Carboxylcytosine
journal, July 2011
- Ito, S.; Shen, L.; Dai, Q.
- Science, Vol. 333, Issue 6047
M. Sma l is an N4-methylcytosine specific DNA-methylase
journal, January 1990
- Klimašauskas, Saulius; Steponavičiene, Dana; Maneliene, Zita
- Nucleic Acids Research, Vol. 18, Issue 22
Highly Integrated Single-Base Resolution Maps of the Epigenome in Arabidopsis
journal, May 2008
- Lister, Ryan; O'Malley, Ronan C.; Tonti-Filippini, Julian
- Cell, Vol. 133, Issue 3
WebLogo: A Sequence Logo Generator
journal, May 2004
- Crooks, Gavin E.; Hon, Gary; Chandonia, John-Marc
- Genome Research, Vol. 14, Issue 6, p. 1188-1190
Methylation by a Unique α-class N4-Cytosine Methyltransferase Is Required for DNA Transformation of Caldicellulosiruptor bescii DSM6725
journal, August 2012
- Chung, Daehwan; Farkas, Joel; Huddleston, Jennifer R.
- PLoS ONE, Vol. 7, Issue 8
Overcoming restriction as a barrier to DNA transformation in Caldicellulosiruptor species results in efficient marker replacement
journal, January 2013
- Chung, Daehwan; Farkas, Joel; Westpheling, Janet
- Biotechnology for Biofuels, Vol. 6, Issue 1
Bacterial genetics: past achievements, present state of the field, and future challenges
journal, April 2008
- Schweizer, Herbert P.
- BioTechniques, Vol. 44, Issue 5
Improved growth media and culture techniques for genetic analysis and assessment of biomass utilization by Caldicellulosiruptor bescii
journal, November 2012
- Farkas, Joel; Chung, Daehwan; Cha, Minseok
- Journal of Industrial Microbiology & Biotechnology, Vol. 40, Issue 1, p. 41-49
Transgenerational Epigenetic Instability Is a Source of Novel Methylation Variants
journal, September 2011
- Schmitz, R. J.; Schultz, M. D.; Lewsey, M. G.
- Science, Vol. 334, Issue 6054
Direct detection of DNA methylation during single-molecule, real-time sequencing
journal, May 2010
- Flusberg, Benjamin A.; Webster, Dale R.; Lee, Jessica H.
- Nature Methods, Vol. 7, Issue 6
Entering the era of bacterial epigenomics with single molecule real time DNA sequencing
journal, April 2013
- Davis, Brigid M.; Chao, Michael C.; Waldor, Matthew K.
- Current Opinion in Microbiology, Vol. 16, Issue 2
Functional Analysis of BamHI DNA Cytosine-N4 Methyltransferase
journal, January 2003
- Lindstrom, William M.; Malygin, Ernst G.; Ovechkina, Lidiya G.
- Journal of Molecular Biology, Vol. 325, Issue 4
Tet Proteins Can Convert 5-Methylcytosine to 5-Formylcytosine and 5-Carboxylcytosine
text, January 2011
- C., Wu, S.; Y., Zhang,; B., Collins, L.
- The University of North Carolina at Chapel Hill University Libraries
Bisulfite Sequencing Protocol Displays both 5-Methylcytosine and N4-Methylcytosine
journal, June 1999
- Vilkaitis, Giedrius; Klimašauskas, Saulius
- Analytical Biochemistry, Vol. 271, Issue 1
Identification and characterization of CbeI, a novel thermostable restriction enzyme from Caldicellulosiruptor bescii DSM 6725 and a member of a new subfamily of HaeIII-like enzymes
journal, May 2011
- Chung, Dae-Hwan; Huddleston, Jennifer R.; Farkas, Joel
- Journal of Industrial Microbiology & Biotechnology, Vol. 38, Issue 11
Entering the era of bacterial epigenomics with single molecule real time DNA sequencing
journal, April 2013
- Davis, Brigid M.; Chao, Michael C.; Waldor, Matthew K.
- Current Opinion in Microbiology, Vol. 16, Issue 2
‘Leveling’ the playing field for analyses of single-base resolution DNA methylomes
journal, December 2012
- Schultz, Matthew D.; Schmitz, Robert J.; Ecker, Joseph R.
- Trends in Genetics, Vol. 28, Issue 12
Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning
journal, February 2008
- Cokus, Shawn J.; Feng, Suhua; Zhang, Xiaoyu
- Nature, Vol. 452, Issue 7184
Human DNA methylomes at base resolution show widespread epigenomic differences
journal, October 2009
- Lister, Ryan; Pelizzola, Mattia; Dowen, Robert H.
- Nature, Vol. 462, Issue 7271
Genomics of DNA cytosine methylation in Escherichia coli reveals its role in stationary phase transcription
journal, January 2012
- Kahramanoglou, Christina; Prieto, Ana I.; Khedkar, Supriya
- Nature Communications, Vol. 3, Issue 1
Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine
journal, November 2012
- Yu, Miao; Hon, Gary C.; Szulwach, Keith E.
- Nature Protocols, Vol. 7, Issue 12
MethylC-seq library preparation for base-resolution whole-genome bisulfite sequencing
journal, February 2015
- Urich, Mark A.; Nery, Joseph R.; Lister, Ryan
- Nature Protocols, Vol. 10, Issue 3
Mechanisms of, and Barriers to, Horizontal Gene Transfer between Bacteria
journal, September 2005
- Thomas, Christopher M.; Nielsen, Kaare M.
- Nature Reviews Microbiology, Vol. 3, Issue 9
Cleavage of methylated CCCGGG sequences containing either N4-methylcytosine or 5-methytcytosine with Mspl, Hpall, Smal, Xmal and Cfr9I restriction endonudeases
journal, January 1987
- Butkus, V.; Petrauskiene, L.; Maneliene, Z.
- Nucleic Acids Research, Vol. 15, Issue 17
M. Sma l is an N4-methylcytosine specific DNA-methylase
journal, January 1990
- Klimašauskas, Saulius; Steponavičiene, Dana; Maneliene, Zita
- Nucleic Acids Research, Vol. 18, Issue 22
Highlights of the DNA cutters: a short history of the restriction enzymes
journal, October 2013
- Loenen, Wil A. M.; Dryden, David T. F.; Raleigh, Elisabeth A.
- Nucleic Acids Research, Vol. 42, Issue 1
Epigenome-wide inheritance of cytosine methylation variants in a recombinant inbred population
journal, June 2013
- Schmitz, R. J.; He, Y.; Valdes-Lopez, O.
- Genome Research, Vol. 23, Issue 10
Tet-Mediated Formation of 5-Carboxylcytosine and Its Excision by TDG in Mammalian DNA
journal, August 2011
- He, Y. -F.; Li, B. -Z.; Li, Z.
- Science, Vol. 333, Issue 6047
Transgenerational Epigenetic Instability Is a Source of Novel Methylation Variants
journal, September 2011
- Schmitz, R. J.; Schultz, M. D.; Lewsey, M. G.
- Science, Vol. 334, Issue 6054
Enhanced 5-methylcytosine detection in single-molecule, real-time sequencing via Tet1 oxidation
journal, January 2013
- Clark, Tyson A.; Lu, Xingyu; Luong, Khai
- BMC Biology, Vol. 11, Issue 1
Overcoming restriction as a barrier to DNA transformation in Caldicellulosiruptor species results in efficient marker replacement
journal, January 2013
- Chung, Daehwan; Farkas, Joel; Westpheling, Janet
- Biotechnology for Biofuels, Vol. 6, Issue 1
Bacterial genetics: past achievements, present state of the field, and future challenges
journal, April 2008
- Schweizer, Herbert P.
- BioTechniques, Vol. 44, Issue 5
Works referencing / citing this record:
A Small RNA Transforms the Multidrug Resistance of Pseudomonas aeruginosa to Drug Susceptibility
journal, June 2019
- Law, Carmen Oi Kwan; Huang, Chuan; Pan, Qing
- Molecular Therapy - Nucleic Acids, Vol. 16
Meta-4mCpred: A Sequence-Based Meta-Predictor for Accurate DNA 4mC Site Prediction Using Effective Feature Representation
journal, June 2019
- Manavalan, Balachandran; Basith, Shaherin; Shin, Tae Hwan
- Molecular Therapy - Nucleic Acids, Vol. 16
Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNA
journal, June 2019
- O’Brown, Zach K.; Boulias, Konstantinos; Wang, Jie
- BMC Genomics, Vol. 20, Issue 1
A Deep Neural Network for Identifying DNA N4-Methylcytosine Sites
journal, March 2020
- Zeng, Feng; Fang, Guanyun; Yao, Lan
- Frontiers in Genetics, Vol. 11
N4-Cytosine DNA Methylation Is Involved in the Maintenance of Genomic Stability in Deinococcus radiodurans
journal, August 2019
- Li, Shengjie; Cai, Jianling; Lu, Huizhi
- Frontiers in Microbiology, Vol. 10