(A) Methylation atlas, based on Illumina 450K arrays, composed of 32 tissues and sorted cells (columns). For each immune cell type we chose the top 10 CpGs that are hypomethylated (yellow) in the specific immune cell type and hypermethylated (blue) in other tissues and cells. This yielded 70 cell-specific CpG sites (rows) for seven different immune cell subtypes – B-cells, CD8 cytotoxic T-cells, CD3 T-cells, regulatory T-cells, eosinophils, monocytes, and neutrophils. (B) Methylation patterns of 17 loci, selected from the 70 shown in panel A, based on the presence of multiple adjacent hypomethylated CpGs within an amplicon of up to 160 bp. Each methylation marker (columns) was assessed using genomic DNA from 19 different tissues and cell types (rows). All 17 markers were amplified in one multiplex PCR. Shades of gray represent the percentage of fully unmethylated molecules from the indicated marker in DNA from the indicated cell type. (C) Spike-in experiments assessing assay sensitivity. Human leukocyte DNA was mixed with DNA from HEK-293 cells (human embryonic kidney cells) in the indicated proportions. Colored lines show the inferred percentage of DNA from the indicated immune cell type in the mixture, as a function of the percentage of leukocyte DNA in the mixture. The percentage of DNA from each immune cell type was calculated using markers specific to neutrophils (NEUT1, NEUT2, NEUT3), monocytes (MONO1, MONO2), eosinophils (EOSI2, EOSI3), B-cells (B-CELL1, B-CELL2, B-CELL3), CD3 T-cells (T-CELL1, T-CELL2), CD8 cytotoxic T-cells (CD8A, CD8B), and regulatory T-cells (TREG1, TREG2).