Bisulfite sequencing validation for MethylCap-Seq. (A) Sequence reads for the Frag_01 (genomic region located at position Ch2: 119,331,343 to 119,331,692) in a tumor (2214 T) and matching lung tissue (2213 N), plotted in the Genome Browser, showing the distribution of the 25 CpGs contained in the fragment highlighted in green. Dashed lines represent the highest number of reads in 2213 N and 2214 T. (B) Methylation status of each CpG in all 36 individually sequenced clones in the same samples and fragment shown in Figure 4A. The middle row represents the average of methylation in all clones per CpG position. (C) Average of methylation of all clones sequenced per patient in each fragment (M: control totally methylated DNA; N:lung; T:tumor; U:control totally unmethylated DNA). (D) Average of the methylation status of the sum of all clones, grouped per histological subtype; comparison betweenadenocarcinoma and squamous cell carcinoma in all fragments were statistically significant. (E) Correlation between normalized methylation signals from MethylCap-seq and CpG methylation from bisulfite sequence. Different regions are shown in different colors; lung samples are marked by dots and tumors by stars. Pearson’s correlation coefficient is denoted above the linear regression curve. *P≤0.001.