Cytosine DNA methylation is important in regulating gene expression and in silencing transposons and other repetitive sequences1,2. Recent genomic studies in Arabidopsis thaliana have revealed that many endogenous genes are methylated either within their promoters or within their transcribed regions, and that gene methylation is highly correlated with transcription levels3,4,5. However, plants have different types of methylation controlled by different genetic pathways, and detailed information on the methylation status of each cytosine in any given genome is lacking. To this end, we generated a map at single-base-pair resolution of methylated cytosines for Arabidopsis, by combining bisulphite treatment of genomic DNA with ultra-high-throughput sequencing using the Illumina 1G Genome Analyser and Solexa sequencing technology6. This approach, termed BS-Seq, unlike previous microarray-based methods, allows one to sensitively measure cytosine methylation on a genome-wide scale within specific sequence contexts. Here we describe methylation on previously inaccessible components of the genome and analyse the DNA methylation sequence composition and distribution. We also describe the effect of various DNA methylation mutants on genome-wide methylation patterns, and demonstrate that our newly developed library construction and computational methods can be applied to large genomes such as that of mouse.
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We thank Y. Bernatavichute for nuclear DNA isolation protocols, A. Clarke for providing embryonic stem cell DNA, A. Girard and G. Hannon for providing mouse germ cell DNA, J. Hetzel for technical assistance, and C. F. Li for assistance with rDNA annotation. S.F. is a Howard Hughes Medical Institute Fellow of the Life Science Research Foundation. X.Z. was supported by a fellowship from the Jonsson Cancer Center Foundation. S.E.J. is an investigator of the Howard Hughes Medical Institute. This work was supported in part by grants from the NSF Plant Genome Research Program and the NIH, and some aspects of the work were performed in the UCLA DNA Microarray Facility.
Author Contributions S.J.C. developed computational methods for mapping and base-calling. S.F. designed and created DNA libraries and performed all molecular biology experiments. S.F., Z.C., B.M. and S.F.N. sequenced the libraries. M.P., S.J.C., S.F. and S.E.J. analysed data. S.E.J. and M.P. designed and directed the study. X.Z., C.D.H. and S.P. assisted in the design of experiments. S.F. and S.J.C. wrote the manuscript.
C. Haudenschild is an employee of Illumina Inc., the manufacturer of the high throughput sequencing system used in this work.
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Cokus, S., Feng, S., Zhang, X. et al. Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature 452, 215–219 (2008). https://doi.org/10.1038/nature06745
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