<p>DNA modifications drive aging, neurodegeneration, carcinogenesis and chemotherapy drug action. Accurate mapping of diverse DNA modifications with single-nucleotide precision in complex genomes remains challenging. We upgraded click-code-seq, a click-chemistry-aided DNA-modification mapping strategy, to enable its first application for sequencing oxidation and depurination in the human genome. We developed a companion fluorescence assay, click-fluoro-quant, to rapidly quantify common DNA modifications and novel adaptors to minimize false positives and assess modification frequency. We uncovered that endogenous DNA oxidation in a human cell line mirrors cancer mutational signatures linked to oxidative stress. The chemotherapy drug irofulven preferentially induces depurination in ApA dimers and promoters. Notably, oxidized guanines and apurinic sites, both irofulven induced and endogenous, are depleted in gene transcribed strands, with the strand bias increasing with gene expression. This work substantially advances click-code-seq for deciphering the impacts of key modifications in human DNA on cellular physiology and toxicological responses.</p><p></p>