title: 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.
authors.affiliation: Department of Biomedical Engineering, Yale University, New Haven, CT 06520, USA.
authors.forename: Lin
authors.initials: L
authors.lastname: Han
authors.affiliation: Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, MA 02115, USA.|Department of Biostatistics, Harvard T. H. Chan School of Public Health, Boston, MA 02215, USA.
authors.forename: Hua-Jun
authors.initials: HJ
authors.lastname: Wu
authors.affiliation: Department of Genetics, Yale School of Medicine, New Haven, CT 06520, USA.|Department of Cell Biology, Second Military Medical University, Shanghai 200433, China.
authors.forename: Haiying
authors.initials: H
authors.lastname: Zhu
authors.affiliation: Department of Genetics, Yale Stem Cell Center, Yale School of Medicine, 10 Amistad, 201B, New Haven, CT 06520, USA.
authors.forename: Kun-Yong
authors.initials: KY
authors.lastname: Kim
authors.affiliation: Department of Genetics, Yale School of Medicine, New Haven, CT 06520, USA.
authors.forename: Sadie L
authors.initials: SL
authors.lastname: Marjani
authors.affiliation: Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, MA 02115, USA.|Department of Biostatistics, Harvard T. H. Chan School of Public Health, Boston, MA 02215, USA.
authors.forename: Markus
authors.initials: M
authors.lastname: Riester
authors.affiliation: Department of Genetics, Stanford University, Palo Alto, CA 94305, USA.
authors.forename: Ghia
authors.initials: G
authors.lastname: Euskirchen
authors.affiliation: Department of Genetics, Yale School of Medicine, New Haven, CT 06520, USA.|Department of Biomedical Engineering, Yale University, New Haven, CT 06520, USA.|Department of Cell Biology, Second Military Medical University, Shanghai 200433, China.
authors.forename: Xiaoyuan
authors.initials: X
authors.lastname: Zi
authors.affiliation: Department of Genetics, Yale School of Medicine, New Haven, CT 06520, USA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Yang
authors.affiliation: Department of Biomedical Engineering, Yale University, New Haven, CT 06520, USA.
authors.forename: Jasper
authors.initials: J
authors.lastname: Han
authors.affiliation: Department of Genetics, Stanford University, Palo Alto, CA 94305, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Snyder
authors.affiliation: Department of Genetics, Yale Stem Cell Center, Yale School of Medicine, 10 Amistad, 201B, New Haven, CT 06520, USA.
authors.forename: In-Hyun
authors.initials: IH
authors.lastname: Park
authors.affiliation: Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, MA 02115, USA.|Department of Biostatistics, Harvard T. H. Chan School of Public Health, Boston, MA 02215, USA.
authors.forename: Rafael
authors.initials: R
authors.lastname: Irizarry
authors.affiliation: Department of Genetics, Yale School of Medicine, New Haven, CT 06520, USA.
authors.forename: Sherman M
authors.initials: SM
authors.lastname: Weissman
authors.affiliation: Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, MA 02115, USA.|Department of Biostatistics, Harvard T. H. Chan School of Public Health, Boston, MA 02215, USA.
authors.forename: Franziska
authors.initials: F
authors.lastname: Michor
authors.affiliation: Department of Biomedical Engineering, Yale University, New Haven, CT 06520, USA.
authors.forename: Rong
authors.initials: R
authors.lastname: Fan
authors.affiliation: Department of Genetics, Yale School of Medicine, New Haven, CT 06520, USA.|Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, Guangzhou, China.|Guangdong Key Laboratory of Biochip Technology, Southern Medical University, Guangzhou 510515, Guangdong, China.
authors.forename: Xinghua
authors.initials: X
authors.lastname: Pan
mesh_terms: D002460:Cell Line; D045744:Cell Line, Tumor; D002874:Chromosome Mapping; D018899:CpG Islands; D019175:DNA Methylation; D004262:DNA Restriction Enzymes / Q000737:chemistry; D044127:Epigenesis, Genetic; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D014644:Genetic Variation; D015894:Genome, Human; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D020014:K562 Cells; D008214:Lymphocytes / Q000166:cytology / Q000378:metabolism; D011401:Promoter Regions, Genetic; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D004262:DNA Restriction Enzymes