Comparative analysis of single-stranded and non-canonical DNA formation in human and other ape cells with telomere-to-telomere genomes.

Non-canonical (non-B) DNA secondary structures, e.g., G-quadruplexes and triplex DNA, are mutation hotspots and genome regulators contributing to disease and evolution. Yet they remain uncharacterized in complete genomes in vivo. Here we exploited the fact that many non-B DNA structures form single-stranded DNA (ssDNA). Using permanganate/S1 footprinting across 14 cell lines, we generated ssDNA profiles for human and six non-human ape telomere-to-telomere (T2T) genomes. Newly resolved satellite arrays-e.g., at ribosomal DNA and centromeres-displayed high ssDNA levels, implicating non-B DNA in satellite expansion and function. Hidden Markov Models applied to our ssDNA data revealed active genomic domains with specific functions-e.g., replication, transcription, or recombination-each enriched in particular non-B DNA types. Human-specific ssDNA domains correlated with nervous system genes, whereas cancer and embryonic cells showed increased ssDNA in transposable elements. Our ssDNA analysis across ape T2T genomes uncovered conserved and species-specific DNA structural dynamics central to genome regulation.