High definition profiling of mammalian DNA methylation by array capture and single molecule bisulfite sequencing

Abstract

DNA methylation stabilizes developmentally programmed gene expression states. Aberrant methylation is associated with disease progression and is a common feature of cancer genomes. Presently, few methods enable quantitative, largescale, single-base resolution mapping of DNA methylation states in desired regions of a complex mammalian genome. Here, we present an approach that combines array-based hybrid selection and massively parallel bisulfite sequencing to profile DNA methylation in genomic regions spanning hundreds of thousands of bases. This single molecule strategy enables methylation variable positions to be quantitatively examined with high sampling precision. Using bisulfite capture, we assessed methylation patterns across 324 randomly selected CpG islands (CGI) representing more than 25,000 CpG sites. A single lane of Illumina sequencing permitted methylation states to be definitively called for >90% of target sties. The accuracy of the hybrid-selection approach was verified using conventional bisulfite capillary sequencing of cloned PCR products amplified from a subset of the selected regions. This confirmed that even partially methylated states could be successfully called. A comparison of human primary and cancer cells revealed multiple differentially methylated regions. More than 25% of islands showed complex methylation patterns either with partial methylation states defining the entire CGI or with contrasting methylation states appearing in specific regional blocks within the island. We observed that transitions in methylation state often correlate with genomic landmarks, including transcriptional start sites and intron-exon junctions. Methylation, along with specific histone marks, was enriched in exonic regions, suggesting that chromatin states can foreshadow the content of mature mRNAs.

Next Generation Sequencing - Advances, Applications and Challenges

“…Whilst being able to detect changes in methylation is interesting, it is more interesting, and indeed more likely to be of functional importance, if this change associates with other detectable biological signals. For example, the potential of associating a methylation change with a corresponding change in transcription of a particular splice variant [50][51][52] from RNA-seq, or with an increase in binding of a specific transcription factor using ChIP-seq data [53].…”

Great expectations: using massively parallel sequencing to solve inherited disorders

“…Region-specific captures have also been employed for identifying somatic mutations on the X chromosome in childhood T-cell acute lymphoblastic leukemia (OMIM 613065) where there is an increased incidence of affected males [22], refining the break points of genomic rearrangements [23] and searching for second-hit recessive mutations within heterozygous deletions [24]. Epigenetic variation has also been mapped and even quantified by specifically targeting CpG islands and performing bisulfite sequencing [25], although interpreting these data must be viewed in light of the original tissue that the DNA was extracted from and whether it is relevant to the disease phenotype.…”

Diverse Domains of (Cytosine-5)-DNA Methyltransferases: Structural and Functional Characterization

“…Distribution of methylated sequences in a genome correlates with the presence of H3K36m3 [126,127]. DNA methylation and H3K36m3 serve as marks for histone deacetylation and the following gene suppression [128].…”