Significance
H-DNA is a non–B-form DNA structure containing intramolecular triplex and single-stranded DNA (ssDNA) regions. H-DNA–forming motifs include polypyrimidine•polypurine mirror repeat sequences, which occur frequently in eukaryotic genomes. These motifs have biological impacts on genome stability and processes such as replication and transcription, but their non-B DNA-forming potentials are not fully understood. Here, we show that the triplex-forming potential of H-DNA motifs in the mouse genome can be evaluated by a deep-sequencing technology that uses the ssDNA-specific nuclease S1 to detect ssDNA. It is currently unclear whether the H-DNA detected formed in vitro or was already present in vivo. Nevertheless, this study provides an approach to unveiling structural features of intramolecular triplexes genome-wide at high spatial resolution.