Detecting replication-associated single-stranded DNA

Single-stranded DNA (ssDNA) is used as a readout of replication stress, yet it arises in several contexts. It is a normal intermediate of DNA replication but can accumulate and persist after replication challenges and is also generated by replication-independent processes. These structures differ in strand origin, architecture, biological consequence, and therapeutic response, so ssDNA detection alone rarely identifies the underlying molecular intermediate or how it was generated. Here, we describe the principal sources of replication-associated ssDNA and review how different methods capture distinct aspects of these intermediates, including ssDNA exposure and accessibility, DNA termini, and their broader structural and replication context. As these features are shared across intermediates, individual readouts often capture multiple structures rather than uniquely identifying one. Reliable interpretation therefore requires integrating complementary signals with experimental perturbations and replication context. Future methods combining these features on individual DNA molecules may enable direct molecular classification of replication intermediates.