Project description:Background: Epigenome-wide association studies (EWAS) have been widely applied to identify methylation CpG sites associated with human disease. To date, the Infinium Methylation EPIC array (EPIC) is commonly used for high-throughput DNA methylation profiling. However, the EPIC array covers only 30% of the human methylome. Methylation Capture bisulfite sequencing (MC-seq) captures target regions of methylome and has advantages of extensive coverage in the methylome at an affordable price. Methods: Epigenome-wide DNA methylation in four peripheral blood mononuclear cell samples was profiled by using SureSelectXT Methyl-Seq for MC-seq and EPIC platforms separately. CpG site-based reproducibility of MC-seq was assessed with DNA sample inputs ranging in quantity of high (> 1000ng), medium (300-1000ng), and low (150ng-300ng). To compare the performance of MC-seq and the EPIC arrays, we conducted a Pearson correlation and methylation value difference at each CpG site that was detected by both MC-seq and EPIC. We compared the percentage and counts in each CpG island and gene annotation between MC-seq and the EPIC array. Results: After quality control, an average of 3,708,550 CpG sites per sample was detected by MC-seq with DNA quantity >1000ng. Reproducibility of MC-seq detected CpG sites was high with strong correlation estimates for CpG methylation among samples with high, medium, and low DNA inputs (r > 0.96). The EPIC array captured an average of 846,464 CpG sites per sample. Compared with the EPIC array, MC-seq detected more CpGs in coding regions and CpG islands. Among the 472,540 CpG sites captured by both platforms, methylation of a majority of CpG sites was highly correlated in the same sample (r: 0.98~0.99). However, methylation for a small proportion of CpGs (N=235) differed significantly between the two platforms, with differences in beta values of greater than 0.5. Conclusions: Our results show that MC-seq is an efficient and reliable platform for methylome profiling with a broader coverage of the methylome than the array-based platform. Although methylation measurements in majority of CpGs are highly correlated, a number of CpG sites show large discrepancy between the two platforms, which warrants further investigation and needs cautious interpretation.

Project description:Here we describe a method for whole genome bisulfite sequencing in very small cell populations (µWGBS) and in single cells (scWGBS). Our method is optimized for profiling DNA methylation in many samples at low coverage, and we developed a bioinformatic method that uses collections of single-cell methylomes to infer cell state dynamics. Using these technological advances, we studied epigenetic cell state dynamics in three in vitro models of cellular differentiation and pluripotency and identified patterns of epigenetic remodeling and cell-to-cell heterogeneity. 1-cell or 4-cell samples were collected from a variety of human and mouse cell lines (KBM7, CCE, K562, 32D, HL60). We also included negative controls (no cells), and cross controls (cells of one species aligned to the other). K562 cells were treated with 5-azacytidine in a final concentration of 1µM to induce demethylation. HL60 cells were treated with 1α,25-dihydroxyvitamin D3 in a final concentration of 10nM to induce differentiation. Naïve pluripotency of CCE ES cells was induced by culturing the cells in ESGRO-2i medium with the addition of 1% FCS. Differentiation of CCE ES cells was induced by treating the cells with all-trans retinoic acid (ATRA) in a final concentration of 0,3µM every third day along with complete LIF-free medium exchange. Embryoid body formation from CCE ES cells was induced by hanging-drop culture for 5 days, followed by 9 days on gelatinized dishes (see treatment annotation). Each experiment, with treated and untreated cells, was performed in multiple biological replicates. We then measured DNA methylation in the individual 1-cell or 4-cell experiments with bisulfite treatment and high-throughput sequencing. For sequencing, most experiments pooled a 4-cell experiment from one species with a 1-cell experiment from the other, but some replicates were sequenced as a single cell (indicated with SC in the replicate annotation). Two samples (marked as “deep”) were sequenced much deeper than the others. See Farlik et al. (2015) Cell Reports for complete study details.

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High-coverage allele-resolved single-cell DNA methylation profiling by scDEEP-mC reveals cell lineage, X-inactivation state, and replication dynamics

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