Finding the fifth base: genome-wide sequencing of cytosine methylation

Abstract

Complete sequences of myriad eukaryotic genomes, including several human genomes, are now available, and recent dramatic developments in DNA sequencing technology are opening the floodgates to vast volumes of sequence data. Yet, despite knowing for several decades that a significant proportion of cytosines in the genomes of plants and animals are present in the form of methylcytosine, until very recently the precise locations of these modified bases have never been accurately mapped throughout a eukaryotic genome. Advanced "next-generation" DNA sequencing technologies are now enabling the global mapping of this epigenetic modification at single-base resolution, providing new insights into the regulation and dynamics of DNA methylation in genomes.

Figure 1.

Techniques for genome-wide sequencing of cytosine methylation sites. Three techniques used recently to generate bisulfite (BS) sequencing libraries compatible with next-generation sequencing are depicted. (A) MethylC-seq (Lister et al. 2008). Double-stranded universal adapter sequences in which all cytosines are methylated are ligated to fragmented genomic DNA. Sodium bisulfite treatment converts unmethylated cytosines to thymine, after which library yield enrichment by PCR with primers complementary to the universal adapter sequences produces the final library that can be sequenced. (B) BS-seq (Cokus et al. 2008). Ligation of a first set of double-stranded adaptors that contained methylated adenine bases within DpnI restriction sites close to the site of ligation with genomic DNA. After BS conversion, PCR is performed using primers complementary to the converted adapter sequences, yielding double-stranded DNA that is digested with DpnI to remove only the first adapter set. Sequencing adapters are subsequently ligated to the double-stranded BS-converted genomic DNA fragments, and PCR with primers complementary to the adapters performed to yield a sequencing library. (C) Reduced representation BS sequencing (RRBS) (Meissner et al. 2008). Genomic DNA is first digested by the methylation-insensitive MspI restriction enzyme, which cleaves the phosphodiester bond upstream of the CpG dinuclotide in its CCGG recognition element. Digested DNA is then separated by gel electrophoresis, and one or more specific size fractions are selected. The size-selected DNA is then end repaired, ligated to double-stranded methylated sequencing adapters (as described above for MethylC-seq), BS converted, and amplified by PCR with primers complementary to the adapter sequences.

Figure 2.

Techniques for enrichment of methylated or target regions prior to BS sequencing. Five approaches that may be used to reduce the complexity of a sample before BS conversion and next-generation sequencing are depicted, targeting methylated regions or select target sequences. (A) MeDIP. Methylated fragments of genomic DNA are immunoprecipitated with an anti-5-methylcytosine antibody. Purified, immunoprecipitated DNA is ligated to double-stranded universal adapter sequences in which all cytosines are methylated. Sodium bisulfite treatment converts unmethylated cytosines to thymine, after which library yield enrichment by PCR with primers complementary to the universal adapter sequences produces the final library that can be sequenced. (B) MBD. Methylated fragments of genomic DNA are isolated from a complex mix of fragmented genomic DNA with a methyl binding domain protein, after which adapter ligation, BS conversion, and PCR enrichment are performed as in A. (C) Microarray capture. Target sequences within a complex mix of fragmented genomic DNA are captured by hybridization to specific oligonucleotides on the surface of a microarray. Following isolation of the hybridized genomic DNA, adapter ligation, BS conversion, and PCR enrichment are performed as in A. (D) Capture in solution. Specific target regions within a mix of fragmented genomic DNA are captured by hybridization to specific oligonucleotides attached to beads in solution. Following isolation of the hybridized genomic DNA, adapter ligation, BS conversion, and PCR enrichment are performed as in A. (E) Molecular inversion probe capture. Fragmented genomic DNA is BS converted, after which molecular inversion probes are added that are designed to hybridize to specific target sequences after conversion. Polymerization primed by the 3′ end of the molecular inversion probe followed by ligation generates a circular molecule that contains the target sequence and is not digested by subsequent exonuclease treatment. PCR using primers that hybridize to the ends of the molecular inversion probes allows amplification of the target region, to which double-stranded universal adapter sequences are ligated to produce a library that is sufficient for next-generation sequencing.