Single-cell mapping of chromosome breaks identifies multiple fragile site classes with distinct DNA replication timing landscapes.

Common fragile sites (CFSs) are genomic regions susceptible to chromosome breaks. However, because they were identified using cell populations, low-frequency fragile sites (non-CFSs) could have been overlooked. Here we perform single-cell genome-wide mapping and classification of aphidicolin-induced chromosome breaks. In human osteosarcoma cells, 42% of breaks exhibit features of CFSs. The remaining 58% are non-CFSs that are 'rare' and replicate throughout the S-phase. Among non-CFSs, we identify early-S replicating breaks associated with transcription-replication conflicts, as well as two additional break classes. One is dependent on transcription and coincides with early-to-late-S replication timing (RT) transition regions, while another class exhibits late-S RT with its frequency increasing upon transcription inhibition. Intriguingly, we find that different RT landscapes are associated with distinct aphidicolin-induced RT behavior and break classes. Our data reveal the frequencies, RT landscapes, and mechanistic differences among distinct break classes, providing a comprehensive view of genomic regions prone to breakage under replication stress.