Fig. 2

BSP and NBSP of plasmid and genomic DNA standards. Representative sequencing chromatograms of plasmid DNA standards characterized by 25 % (a) and 75 % (b) CGI methylation (see Methods). Each mixture was PCR amplified with the 5’-end tailed primers for miR-200c/miR-141 locus and the amplicons were sequenced using the Tail1 as a sequencing primer. Left panels depict 6 out of 14 CpG analyzed (indicated by gray arrows), while the right panels show the chromatograms relative to the Tail2-200c-R primer for miR-200c/miR-141 locus. C and T used to calculate the NF in the NBSP are highlighted by black and white arrows, respectively. c-d Bland–Altman plots of plasmid DNA standards (c) and genomic DNA standards (d) show the extent to which observed and expected methylation values of DNA standards agree. Methylation was evaluated by BSP (c and d, left panels) or NBSP (c and d, right panels). The solid lines represent the mean percentage difference between observed and expected (Bias) and the dashed lines ±1.96 SD of the mean percentage difference (limits of agreement). Filled circles represent individual measurements