Fig. 2: Validation of SSiNGLe-AP.

a Preparation and application of the spike-ins. The spike-ins were PCR amplified in the presence of dUTP and then either treated with UDG (uracil-DNA glycosylase) to produce AP sites at thymines (Ts) and serve as the positive spike-ins (“P”) or were not treated and serve as the negative spike-ins (“N”). Mammalian DNA samples containing the positive and negative spike-ins were processed by the SSiNGLe-AP procedure to generate the “P-A” samples, “N-A” or unblocked (Fig. 1) controls and used to estimate the background, specificity and precision of the method. b Representative DNA size distribution profiles of NGS libraries made using SSiNGLe-AP with (“+APE1”) or without (“‒APE1”) the APE1 treatment from mammalian DNA containing the positive spike-ins. The portions of the profiles representing the actual genomic material are boxed—the peaks to the left represent primer dimers. c Aggregate plots of relative depths of AP sites (“Methods”) along the length of positive spike-in sequences in the “P-A” or unblocked controls generated by combining sites from both strands of all spike-ins and all technical replicates. d Bar plots indicate the average fractions of each nucleotide occurring in the spike-in sequences (“Background”), as well as in all AP sites and hotspots detected with variable thresholds in the 3 spike-ins of the “P-A” samples (“Methods”). e Normalized relative depth (Methods) of mapped sites from 3 independent technical replicates of positive (“P-A”) and negative (“N-A”) spike-ins are plotted along the length of the “+” strand of the spike-in S1. Zoomed-in windows show two AP site hotspots, marked by arrows, peaking at T nucleotides. f The abundancies of AP sites in the samples treated with MX (methoxyamine), MMS (methyl methanesulfonate) and the corresponding controls are represented as either APL or APF (“Methods”). Data are presented as mean values +/− SD based on 3 biological replicates. Source data are provided as a Source data file.