Fig. 1

Detection of BrdU in human genomic DNA by nanopore sequencing over a wide range of BrdU concentrations. A Plots show two example nanopore sequencing reads from an asynchronous HeLa-S3 cell culture treated with 10 µM BrdU for 20 h. Reads are shown aligned to the human genome with DNAscent calculated BrdU probabilities for each thymidine position. Tick marks are 10 kb. B Frequency distribution of BrdU probabilities at every thymidine position for nanopore sequencing reads from HeLa-S3 cells grown in the indicated range of BrdU concentrations for 20 h. Insert shows the full y-axis for the 50 µM sample. C Two plots of the fraction BrdU incorporated (p ≥ 0.5; independent windows of 290 thymidine positions) for the example nanopore reads in Fig. 1 A. Tick marks are 10 kb. D Frequency distribution of the fraction BrdU incorporated in each window (290 thymidine) for same reads as in Fig. 1B. E Comparison of fraction BrdU in DNA as determined by nanopore sequencing and mass spectrometry from the same DNA samples (as in Fig. 1B, D). Dashed line is y = x. F CpG methylation analysis for the two example reads in Fig. 1 A, C. Top row: Jitter plot of individual methylated CpGs determined by Nanopolish. Middle row: smoothed density of CpG methylation across the two reads. Bottom row: fraction GC in 1-kb windows. G Meta-analysis of fraction BrdU detected by DNAscent at thymidine positions relative to CGI centers for reads from HeLa-S3 cells treated with 0.0 or 50 μM BrdU (as per Fig. 1B, D). Fractions are calculated from 100 -bp windows. Dashed lines (200 bps apart) indicate the minimum bounds of CGIs. CGIs were separated into three groups based on mean methylation level, blue lines show the top third (high methylation), black lines show the bottom third (low methylation)