High-Resolution Chemo-Enzymatic Approaches to Epigenomic Profiling of cfDNA

Analysis of cell-free circulating DNA (cfDNA) proved a promising approach to improve clinical issues related to diagnostics and disease monitoring, although low mutation frequency and the lack of information on the tissue of origin hamper the detection sensitivity. Most lately, epigenomic profiling of cfDNA has emerged as a promising approach to improve such analyses. Modification of cytosine to 5mC in CpG dinucleotides is part of epigenetic regulation of gene function in vertebrates and thus the modification profiles are tissue-specific and can often be associated with the occurrence and progression of human disease. Detection of altered DNA modification profiles could serve as biomarkers of complex diseases, however these studies are hampered by the limitations of existing analytic techniques. We have developed a novel concept in analysis of DNA modification patterns named Tethered Oligonucleotide-Primed sequencing (TOP-seq) that relies on selective read out of genomic regions from covalently tagged individual unmodified CpG sites. This is achieved via a previously unknown feature of DNA polymerases to prime the DNA strand synthesis from an oligonucleotide probe covalently tethered at the target site. Such non-homologous priming produces the adjoining regions for their sequencing and precise genomic mapping. The proposed approach offers single-CpG resolution, avoids whole-genome sequencing and is well suited for large-scale high-resolution studies. Pilot TOP-seq analyses showed an accurate, robust and highly sensitive detection of long-range and gene-level differential methylation among human tissues and neuroblastoma cell types. The method uses ultra-low amounts of input DNA, is flexible with respect to downstream sequencing platforms (Ion Torrent, Illumina), and can be configured for whole-genome profiling or for targeted analysis of selected genomic loci.