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High-Throughput Next Generation Sequencing

Volume 733 of the series Methods in Molecular Biology pp 223-238

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Determining DNA Methylation Profiles Using Sequencing

  • Suhua Feng
  • , Liudmilla Rubbi
  • , Steven E. Jacobsen
  • , Matteo PellegriniAffiliated withDepartment of Molecular, Cell and Developmental Biology, University of California Email author 

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Abstract

Cytosine methylation is an epigenetic mark that has a significant impact on the regulation of transcription and replication of DNA. DNA methylation patterns are highly conserved across cell divisions and are therefore highly heritable. Furthermore, in multicellular organisms, DNA methylation patterning is a key determinant of cellular differentiation and tissue-specific expression patterns. Lastly, DNA demethylases can affect global levels of DNA methylation during specific stages of development. Bisulfite sequencing is considered the gold standard for measuring the methylation state of cytosines. Sodium bisulfite ­converts unmethylated cytosines to uracils (which after PCR are converted to thymines), while leaving methylated cytosines unconverted. By mapping bisulfite treated DNA back to the original reference genome, it is then possible to determine the methylation state of individual cytosines. With the advent of next-generation sequencers during the past few years, it is now possible to determine the methylation state of an entire genome. Here, we describe in detail two protocols for preparing bisulfite treated libraries, which may be sequenced using Illumina GAII sequencers. The first of these uses premethylated adapters, which are not affected by bisulfite treatments, while the second uses a two-stage adapter strategy and does not require premethylation of the adapters. We also describe the specialized protocol for mapping bisulfite converted reads. These approaches allow one to determine the methylation state of each cytosine in the genome.

Key words

DNA methylation Epigenetics Next-generation sequencing Whole-genome methylome