Liang et al. BMC Genomics 2011, 12:231 http://www.biomedcentral.com/1471-2164/12/231 Table 1 Adult T-DMRs Tissue Common Brain unique Heart unique Liver unique Testis unique Total T-unique B + H B + L B + T H + L H + T L + T B + H + L B + H + T B + L + T H + L + T Total multi-tissues Total T-DMRs Total 460 285 822 711 1294 3112 482 12 26 49 47 305 414 175 18 46 1574 4686 CpGi Prom 18 68 223 155 44 490 69 2 1 7 0 3 65 3 0 4 154 644 % 3.9 23.9 27.1 21.8 3.4 15.7 14.3 16.7 3.8 14.3 0.0 1.0 15.7 1.7 0.0 8.7 9.8 13.7 Non-CpGi Prom 230 173 428 428 1215 2244 282 7 25 24 45 268 148 151 15 37 1002 3246 % 50.0 60.7 52.1 60.2 93.9 72.1 58.5 58.3 96.2 49.0 95.7 87.9 35.7 86.3 83.3 80.4 63.7 69.3 Intra-genic CpGi 163 23 115 66 20 224 89 2 0 13 2 27 127 19 2 4 285 509 % 35.4 8.1 14.0 9.3 1.5 7.2 18.5 16.7 0.0 26.5 4.3 8.9 30.7 10.9 11.1 8.7 18.1 10.9 Inter-genic CpGi 49 21 56 62 15 154 42 1 0 5 0 7 74 2 1 1 133 287 Page 4 of 16 % 10.7 7.4 6.8 8.7 1.2 4.9 8.7 8.3 0.0 10.2 0.0 2.3 17.9 1.1 5.6 2.2 8.4 6.1 example, approximately 94% of the testis-specific T-DMRs are in non-CpGi promoter regions, whereas each of the three somatic tissues is no more than 60%. T-DMRs that are present in brain, heart, and liver, but not testis, would include somatic methylation sites. The majority of these (B+H+L) are in CpGi regions (64.3%). The number and distribution of T-DMRs in ES cells ES cells are derived from the inner cell mass of the early blastocyst that gives rise to the embryo. Following fertiliza- tion, the embryonic cells undergo a period of demethyla- tion and make up/form the inner cell mass. Remethylation of somatic tissues is thought to resume following implan- tation [7]. Thus, we compared the methylation status of T-DMRs in E15 tissues with ES cells (Additional file 1). Overall, there are fewer T-DMRs (2750) from the compar- ison of ES cell and E15 tissues than T-DMRs in adult tis- sues (4686, Table 1). We found more unique methylation peaks in ES cells (981) than in E15 tissues of brain (310), heart (131), liver (764), or testis (230). This indicates that there are many sites that are methylated in ES cells but are unmethylated in E15 tissues with most (85.5%) located in non-CpGi promoter regions. T-DMRs in regions outside CpGi regions and promoters The NimbleGen promoter + CpGi array contains a tiling array that covers about 1.9 Mb of the mouse genome. This includes the Hoxa gene cluster on chromosome 6 (423 Kb), the Igf2r imprinted region on chromosome 17 (88 Kb) and imprinted regions on chromosome 7 (1,390 Kb). In these regions it is possible to identify tissue specific methylation peaks that are not associated with CpGi regions or promo- ters. For example, in the KvDMR region on chromosome 7 (Additional file 2), there are non-CpGi and non-promoter methylation peaks in testis and brain that are not present in liver in addition to T-DMRs in CpGi regions. The results shown in Additional file 3a indicate that there is similar dis- tribution of T-DMRs in the tiling regions in CpGi and non- CpGi promoter regions as in the rest of the array-covered genome. However, there are additional methylation peaks within the tiling regions (Additional file 3b) in non-CpGi intragenic regions (15.7%) and in non-CpGi intergenic regions (12.9%). These results indicate that almost 30% of the T-DMRs may be missed by restricting analysis to CpGi and promoter regions. Developmental Stage-Specific Differentially Methylated Regions (DS-DMRs) We used MeDIP to determine the number and location of methylated sites at the different developmental stages within each tissue (Table 2). DS-DMRs are genomic regions that are methylated at one or more developmental stage of a tissue, but unmethylated in at least one stage. It should be noted that DS-DMRs are selected as stage-specific differen- tially methylated regions within a tissue without regard to presence or absence in another tissue. This analysis pro- vides a basis for determining changes in developmental stage specific methylation that occur within a tissue. These changes may reflect active or passive changes in DNA methylation as well as changes in the proportion of specific cell populations. The Pearson Coefficients between the duplicate biological replicates varied somewhat from