Confounding between cellular composition and age at the CpG level. Comparisons between resulting t-statistics for age on DNA methylation levels in Hannum et al.  using (A) naïve (for example, including cell composition estimates as covariates in regression models), (B) two-step Remove Unwanted Variation (RUV), (C) flow-sorted CD4+ T cells and (D) flow-sorted monocytes compared to the effect of age on DNAm in a univariate model. The univariate and naïve models also adjusted for processing plate, which was a very strong confounder. Here, analysis with RUV attenuates the association between DNAm and age. The solid lines indicate the resulting t-statistic cutoff for false discovery rate <5% - no probes were significant at this threshold in the cell sorted data. All panels contain probes present on both the Illumina 450k and 27k (n = 24,692) to facilitate comparisons to age associations in the flow-sorted cellular populations.