title: 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 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.
authors.affiliation: Department of Chemistry and Institute for Biophysical Dynamics, Howard Hughes Medical Institute, The University of Chicago, Chicago, IL 60637, USA.
authors.forename: Miao
authors.initials: M
authors.lastname: Yu
authors.affiliation: Institute of Bioinformatics, The University of Georgia, Athens, GA 30602, USA.
authors.forename: Lexiang
authors.initials: L
authors.lastname: Ji
authors.affiliation: Department of Genetics, The University of Georgia, Athens, GA 30602, USA.
authors.forename: Drexel A
authors.initials: DA
authors.lastname: Neumann
authors.affiliation: Department of Genetics, The University of Georgia, Athens, GA 30602, USA.
authors.forename: Dae-Hwan
authors.initials: DH
authors.lastname: Chung
authors.affiliation: Department of Genetics, The University of Georgia, Athens, GA 30602, USA.
authors.forename: Joseph
authors.initials: J
authors.lastname: Groom
authors.affiliation: Department of Genetics, The University of Georgia, Athens, GA 30602, USA The BioEnergy Science Center, U.S. Department of Energy, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA.
authors.forename: Janet
authors.initials: J
authors.lastname: Westpheling
authors.affiliation: Department of Chemistry and Institute for Biophysical Dynamics, Howard Hughes Medical Institute, The University of Chicago, Chicago, IL 60637, USA chuanhe@uchicago.edu.
authors.forename: Chuan
authors.initials: C
authors.lastname: He
authors.affiliation: Department of Genetics, The University of Georgia, Athens, GA 30602, USA schmitz@uga.edu.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Schmitz
mesh_terms: D044503:5-Methylcytosine / Q000032:analysis; D000818:Animals; D003596:Cytosine / Q000031:analogs & derivatives* / Q000032:analysis; D004269:DNA, Bacterial / Q000737:chemistry*; D004268:DNA-Binding Proteins; D000068536:Bacillota / Q000235:genetics; D016680:Genome, Bacterial; D051379:Mice; D059372:Nucleotide Motifs; D011518:Proto-Oncogene Proteins; D017422:Sequence Analysis, DNA / Q000379:methods*; D013447:Sulfites
chemical_list: D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; D013447:Sulfites; C540839:TET1 protein, mouse; C039052:N(4)-methylcytosine; D044503:5-Methylcytosine; D003596:Cytosine; C042345:hydrogen sulfite