title: Tethered Oligonucleotide-Primed Sequencing, TOP-Seq: A High-Resolution Economical Approach for DNA Epigenome Profiling.
abstract: Modification of CG dinucleotides in DNA is part of epigenetic regulation of gene function in vertebrates and is associated with complex human disease. Bisulfite sequencing permits high-resolution analysis of cytosine modification in mammalian genomes; however, its utility is often limited due to substantial cost. Here, we describe an alternative epigenome profiling approach, named TOP-seq, which is based on covalent tagging of individual unmodified CG sites followed by non-homologous priming of the DNA polymerase action at these sites to directly produce adjoining regions for their sequencing and precise genomic mapping. Pilot TOP-seq analyses of bacterial and human genomes showed a better agreement of TOP-seq with published bisulfite sequencing maps as compared to widely used MBD-seq and MRE-seq and permitted identification of long-range and gene-level differential methylation among human tissues and neuroblastoma cell types. Altogether, we propose an affordable single CG-resolution technique well suited for large-scale epigenome studies.
authors.affiliation: Department of Biological DNA Modification, Institute of Biotechnology, Vilnius University, Vilnius LT-10257, Lithuania.
authors.forename: Zdislav
authors.initials: Z
authors.lastname: Staševskij
authors.affiliation: Department of Biological DNA Modification, Institute of Biotechnology, Vilnius University, Vilnius LT-10257, Lithuania.
authors.forename: Povilas
authors.initials: P
authors.lastname: Gibas
authors.affiliation: Department of Biological DNA Modification, Institute of Biotechnology, Vilnius University, Vilnius LT-10257, Lithuania; Department of Systems Analysis, Institute of Mathematics and Informatics, Vilnius University, Vilnius LT-08663, Lithuania.
authors.forename: Juozas
authors.initials: J
authors.lastname: Gordevičius
authors.affiliation: Department of Biological DNA Modification, Institute of Biotechnology, Vilnius University, Vilnius LT-10257, Lithuania. Electronic address: edita.kriukiene@bti.vu.lt.
authors.forename: Edita
authors.initials: E
authors.lastname: Kriukienė
authors.affiliation: Department of Biological DNA Modification, Institute of Biotechnology, Vilnius University, Vilnius LT-10257, Lithuania. Electronic address: saulius.klimasauskas@bti.vu.lt.
authors.forename: Saulius
authors.initials: S
authors.lastname: Klimašauskas
mesh_terms: D018899:CpG Islands; D019175:DNA Methylation; D017931:DNA Primers / Q000378:metabolism*; D044127:Epigenesis, Genetic; D057890:Epigenomics / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D017931:DNA Primers
keywords: 5-methylcytosine; CpG-specific methylation; DNA polymerase priming; DNA unmethylome profiling; SssI methyltransferase; differential methylation; mTAG labeling; neuroblastoma; next-generation sequencing