Fig 1.
Workflow of the study design in two experiments.
Table 1.
DNA collection protocols used in Experiment 2.
Fig 2.
Correction of systematic biases in DNA methylation profile caused by cold storage using cell-type composition.
A. Quantile-quantile (QQ) plot for the comparison of paired β-values from the 16 individuals between duplicates (Ctrl1 vs. Ctrl2). The genomic inflation factor lambda (median P-value of obs/exp) is shown. B. QQ plot for the comparison of 16 individuals between Ctrl1 and 4°C-24 h conditions. C. QQ plot for the comparison of 16 individuals between Ctrl1 and 4°C-24 h conditions after adjustment for the change in the estimated proportion of granulocytes. D. Differences of cell proportion between conditions (Ctrl2: Ctrl1 vs. Ctrl2; 4°C-24 h: Ctrl1 vs. 4°C-24 h) within the same individual are estimated by the cell-type composition from DNA methylation profiles. CD8T, CD8+ T cells; CD4T, CD4+ T cells; NK, natural killer cells; Bcell, B cells; Mono, monocytes; Gran, granulocytes. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Wilcoxon sighed rank test compared with Ctrl1) E. Gating strategy used to analyze populations of lymphocytes, monocytes, and granulocytes. F. Differences of cell proportion between conditions (Ctrl2: Ctrl1 vs. Ctrl2; 4°C-24 h: Ctrl1 vs. 4°C-24 h) within the same individual are measured by FACS using samples derived another 6 individuals. *, P < 0.05 (Wilcoxon signed-rank test compared with Ctrl1).
Fig 3.
Systematic biases in DNA methylation profile caused by difference of DNA collection protocols.
A. Differences of cell proportion between conditions (Ctrl1 vs. Ctrl2, TMM, BBJ, Hisayama, and JPHC) within the same individual are estimated by the cell-type composition from DNA methylation profiles. CD8T, CD8+ T cells; CD4T, CD4+ T cells; NK, natural killer cells; Bcell, B cells; Mono, monocytes; Gran, granulocytes. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Wilcoxon sighed rank test compared with Ctrl1). B-F. QQ plots for the comparison of 16 individuals between conditions (Ctrl1 vs. Ctrl2, TMM, BBJ, Hisayama, and JPHC) before (brown points) and after adjustment for the change in the estimated proportion of granulocytes (blue points). The genomic inflation factor lambda (median P-value of obs/exp) is shown.