Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells

Abstract

Conventional DNA bisulfite sequencing has been extended to single cell level, but the coverage consistency is insufficient for parallel comparison. Here we report a novel method for genome-wide CpG island (CGI) methylation sequencing for single cells (scCGI-seq), combining methylation-sensitive restriction enzyme digestion and multiple displacement amplification for selective detection of methylated CGIs. We applied this method to analyzing single cells from two types of hematopoietic cells, K562 and GM12878 and small populations of fibroblasts and induced pluripotent stem cells. The method detected 21 798 CGIs (76% of all CGIs) per cell, and the number of CGIs consistently detected from all 16 profiled single cells was 20 864 (72.7%), with 12 961 promoters covered. This coverage represents a substantial improvement over results obtained using single cell reduced representation bisulfite sequencing, with a 66-fold increase in the fraction of consistently profiled CGIs across individual cells. Single cells of the same type were more similar to each other than to other types, but also displayed epigenetic heterogeneity. The method was further validated by comparing the CpG methylation pattern, methylation profile of CGIs/promoters and repeat regions and 41 classes of known regulatory markers to the ENCODE data. Although not every minor methylation differences between cells are detectable, scCGI-seq provides a solid tool for unsupervised stratification of a heterogeneous cell population.

Using single‐cell multiple omics approaches to resolve tumor heterogeneity

“…Comparatively, the scRRBS method only covers 1% CpG sites across the genome, in contrast to 48.4% of CpG sites by scW-GBS. Single-cell methylase assisted bisulfite sequencing (scMAB-seq) and CpG island methylation sequencing for single-cell (scCGI-seq) have also recently been proposed [85,86]. Future comparative studies of these various methods will assist in determining the differences in mapping and cost efficiency.…”

Single-Cell Omics Analyses Enabled by Microchip Technologies

“…Single-cell bisulfite sequencing and reduced-representation bisulfite sequencing were demonstrated using manual protocols with low throughput (68)(69)(70)(71). Alternatively, a methylation-specific enzyme-discrimination method in conjunction with MDA enabled genome-scale profiling of CpG island methylation patterns in single cells (72). These methods can be potentially integrated in microfluidic systems.…”

Mapping chromatin modifications at the single cell level

“…Endonuclease-based sequencing methods avoid random DNA loss and thus improve coverage and efficiency. Genome-wide CpG island (CGI) methylation sequencing for single cells (scCGI-seq) is one such method that enriches for sequences with high CpG content, providing 72.7% CGI coverage per cell (Han et al, 2017) (Fig. 5E).…”

“…Rather, this method relies on the observation that neighboring CpG sites within a 1 kb window have a high probability of sharing the same methylation state (Hui et al, 2018). Hierarchical clustering of CGI methylation revealed by scCGI-seq yielded distinct clusters according to cell type, with greater CGI/promoter hypermethylation and repeat region hypomethylation measured in the cancer cell line K562 compared with the non-cancer cell line GM12878 (Han et al, 2017). This strategy could be extended to mapping 5hmC, although more 5hmC-sensitive restriction enzymes need to be identified to provide greater coverage.…”