title: Repetitive DNA methylome analysis by small-scale and single-cell shotgun bisulfite sequencing.
abstract: Whole-genome shotgun bisulfite sequencing (WG-SBS) is currently the most powerful tool available for understanding genomewide cytosine methylation with single-base resolution; however, the high sequencing cost limits its widespread application, particularly for mammalian genomes. We mapped high- to low-coverage SBS short reads of mouse and human female developing germ cells to consensus sequences of repetitive elements that were multiplied in the respective host genome. This mapping strategy effectively identified active and evolutionarily young retrotransposon subfamilies and centromeric satellite repeats that were resistant to DNA demethylation during the investigated progressive stages of germ cell development. Notably, quantities of only tens of thousands of uniquely mapped reads provided sufficient sensitivity to allow for methylation analyses of multiple retrotransposons and satellite repeats in mice. Furthermore, we produced SBS results from single female murine germ cells by an improved multiplexing and amplification-free SBS method (scPBAT). The scPBAT results quantitatively provided ≥5× sequencing coverage for at least 30 repeats, and the individual methylation patterns detected were similar to the bulk cell-based results. Our single-cell methylome sequencing technique will allow researchers to investigate intergenic methylation characteristics from limited amounts of mammalian cells as well as cells from other organisms with genomic annotations.
authors.affiliation: NODAI Genome Research Center, Tokyo University of Agriculture, Setagaya-ku, Tokyo, 156-8502, Japan.
authors.forename: Hisato
authors.initials: H
authors.lastname: Kobayashi
authors.affiliation: Department of BioScience, Tokyo University of Agriculture, Setagaya-ku, Tokyo, 156-8502, Japan.
authors.forename: Tasuku
authors.initials: T
authors.lastname: Koike
authors.affiliation: Department of BioScience, Tokyo University of Agriculture, Setagaya-ku, Tokyo, 156-8502, Japan.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Sakashita
authors.affiliation: NODAI Genome Research Center, Tokyo University of Agriculture, Setagaya-ku, Tokyo, 156-8502, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Tanaka
authors.affiliation: Department of BioScience, Tokyo University of Agriculture, Setagaya-ku, Tokyo, 156-8502, Japan.
authors.forename: Soichiro
authors.initials: S
authors.lastname: Kumamoto
authors.affiliation: Department of BioScience, Tokyo University of Agriculture, Setagaya-ku, Tokyo, 156-8502, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Kono
mesh_terms: D000818:Animals; D002874:Chromosome Mapping; D019175:DNA Methylation; D005260:Female; D015723:Gene Library; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D010063:Ovum / Q000166:cytology; D012091:Repetitive Sequences, Nucleic Acid; D017422:Sequence Analysis, DNA / Q000379:methods*; D013447:Sulfites
chemical_list: D013447:Sulfites; C042345:hydrogen sulfite