DENT-seq overview and workflow. (A) Chemical structures of the two degenerate nucleotides, dPTP and dKTP, in both of their tautomeric forms enabling their respective activities as universal pyrimidine and purine. Black dotted lines represent hydrogen bonds between nucleotides. (B) During nick translation with only the two degenerate nucleotides, P residues (gold) are inserted across from A (blue) and G (purple) residues while K residues (cyan) are inserted across from C (green) and T (red) residues. (C) Sequencing DNA fragments without nicks or that had nick translation occur with only regular dNTPs will not show a mutational signal (top). Sequencing DNA fragments that underwent nick translation with dPTP and dKTP will show a mutational signal that extends a few bases 3′ of the nick's original location (bottom). (D) Workflow used to perform DENT-seq. Nick translations are performed consecutively with dPTP plus dKTP and then with regular dNTPs plus biotinylated dUTP. Of note, nick translation will move a nick from its original location but will not actually repair the nick. DNA is fragmented and streptavidin-based purification enriches for fragments around the original (pre-nick translation) sites of nicks. After PCR and sequencing, sequence coverage and mutational information allow for sensitive single-nucleotide-resolved and strand-specific identification of nick sites.
