Fig. 1: Schematics of SSiNGLe-AP.

Workflow of the molecular biological part of SSiNGLe-AP starting with fragmentation of genomic dsDNA using DNase, followed by denaturation, blocking of all 3’OH termini of ssDNA molecules using TdT (terminal transferase) and biotin-11-ddCTP, and subsequent affinity capture of 3’-blocked and biotinylated fragments (all symbols are explained in bottom right) that were then subjected to APE1 treatment that releases ssDNA fragments, in which the 3’OH termini correspond to Ns—the nucleotides immediately upstream of the corresponding AP sites. The freed fragments were then subjected to the SSiNGLe for Illumina (SSiNGLe-ILM) procedure starting with tagging the 3’OH termini by polyA-tailing and subsequent NGS library preparation steps. Importantly, DNA molecules in which the 3’ termini were blocked by DNA damage (such as oxidated 3’ ends) cannot be biotinylated and thus did not enter the APE1 treatment step. The spike-ins were added to DNA before denaturation, as shown. To generate the unblocked control, the denatured DNA was used as input into SSiNGLe-ILM, as shown after the addition of spike-ins. See Supplementary Fig. 1 for the description of the analytical parts of SSiNGLe-AP.