Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Jul 5;17(1):150.
doi: 10.1186/s13059-016-1011-3.

Single-cell Genome-Wide Bisulfite Sequencing Uncovers Extensive Heterogeneity in the Mouse Liver Methylome

Free PMC article

Single-cell Genome-Wide Bisulfite Sequencing Uncovers Extensive Heterogeneity in the Mouse Liver Methylome

Silvia Gravina et al. Genome Biol. .
Free PMC article


Background: Transmission fidelity of CpG DNA methylation patterns is not foolproof, with error rates from less than 1 to well over 10 % per CpG site, dependent on preservation of the methylated or unmethylated state and the type of sequence. This suggests a fairly high chance of errors. However, the consequences of such errors in terms of cell-to-cell variation have never been demonstrated by experimentally measuring intra-tissue heterogeneity in an adult organism.

Results: We employ single-cell DNA methylomics to analyze heterogeneity of genome-wide 5-methylcytosine (5mC) patterns within mouse liver. Our results indicate a surprisingly high level of heterogeneity, corresponding to an average epivariation frequency of approximately 3.3 %, with regions containing H3K4me1 being the most variable and promoters and CpG islands the most stable. Our data also indicate that the level of 5mC heterogeneity is dependent on genomic features. We find that non-functional sites such as repeat elements and introns are mostly unstable and potentially functional sites such as gene promoters are mostly stable.

Conclusions: By employing a protocol for whole-genome bisulfite sequencing of single cells, we show that the liver epigenome is highly unstable with an epivariation frequency in DNA methylation patterns of at least two orders of magnitude higher than somatic mutation frequencies.

Keywords: Aging; Epigenetic instability; Epivariations; Single-cell DNA methylomics; Single-cell epigenomics.


Fig. 1
Fig. 1
Global methylation and coverage of single-cell WGBS. a Genome wide 5mC levels and coverage in single fibroblasts (blue) and hepatocytes (red). From outside to inside, the first layer represents 5mC level, the second layer coverage at each CpG site. 5mC levels and coverage were averaged among cells from each group and estimated using 1-Mb non-overlapping sliding windows. b Global 5mC levels at CpG sites for single cells and bulk for the two cell types and two age groups. c Percentage of genomic 3-kb windows containing at least 5 CpG sites in single hepatocytes and fibroblasts. Virtually all qualified windows in the single cells were found to overlap with their bulk samples. Grey, fibroblasts; blue, young hepatocytes; red, old hepatocytes
Fig. 2
Fig. 2
Single-cell WGBS is an accurate and reproducible method for genome-wide 5mC analysis. a 5mC promoter methylation status of 58 liver-specific genes. b Merged single cells have the same methylation pattern as their corresponding bulk. Each comparison is based on 10,000 randomly chosen 3-kb windows indicates the number of single cells sequenced. c Principal component analysis of single cells and bulk shows separate clustering of fibroblasts and hepatocytes (both panels) and hepatocytes from old and hepatocytes from young mice
Fig. 3
Fig. 3
5mC heterogeneity. a Global heterogeneity per cell. Variance value was used to quantify the difference between a cell and its bulk across windows. Raw variance (x-axis) and noise (y-axis) estimated from downsampling bulk to single-cell equivalent were plotted. To test significance of difference in mean variance among groups, P values were obtained by using permutation tests of randomly resampled samples into the two groups for comparison. b Number of differentially methylated windows in fibroblasts and hepatocytes from young and old mice. Differentially methylated window (DMW) frequency was significantly higher in hepatocytes than in fibroblasts (P < 0.001, two-tailed t-test). The slightly higher DMW frequency in hepatocytes from aged mice was not significant. c 5mC heterogeneity in liver is highly dependent on sequence feature. CGI CpG island, LINE long interspersed nuclear element, LTR long terminal repeat, SINE short interspersed nuclear element, UTR untranslated region

Similar articles

See all similar articles

Cited by 28 articles

See all "Cited by" articles


    1. Laird CD, Pleasant ND, Clark AD, Sneeden JL, Hassan KM, Manley NC, Vary JC, Jr., Morgan T, Hansen RS, Stoger R. Hairpin-bisulfite PCR: assessing epigenetic methylation patterns on complementary strands of individual DNA molecules. Proc Natl Acad Sci U S A. 2004;101:204–9. - PMC - PubMed
    1. Modder UI, Sanyal A, Kearns AE, Sibonga JD, Nishihara E, Xu J, O'Malley BW, Ritman EL, Riggs BL, Spelsberg TC, Khosla S. Effects of loss of steroid receptor coactivator-1 on the skeletal response to estrogen in mice. Endocrinology. 2004;145:913–21. - PubMed
    1. Gravina S, Ganapathi S, Vijg J. Single-cell, locus-specific bisulfite sequencing (SLBS) for direct detection of epimutations in DNA methylation patterns. Nucleic Acids Res. 2015;43:e93. doi: 10.1093/nar/gkv366. - DOI - PMC - PubMed
    1. Bahar Halpern K, Tanami S, Landen S, Chapal M, Szlak L, Hutzler A, Nizhberg A, Itzkovitz S. Bursty gene expression in the intact mammalian liver. Mol Cell. 2015;58:147–56. - PMC - PubMed
    1. Smallwood SA, Lee HJ, Angermueller C, Krueger F, Saadeh H, Peat J, Andrews SR, Stegle O, Reik W, Kelsey G. Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity. Nat Methods. 2014;11:817–20. - PMC - PubMed

Publication types


LinkOut - more resources