3.5 Whole-Genome Bisulfite Sequencing
Studies of DNA methylation were initially restricted to localized CpG-rich regions
of the genome [92]. However, several methods now exist which can map DNA
methylation genome-wide. Whole-genome bisulfite sequencing (WGBS) was developed in 1992 by Frommer et al. to measure CpG methylation genome-wide at a
single-base resolution [93]. This method utilizes bisulfite-induced modification of
genomic DNA to convert cytosine to uracil; the latter is converted to thymine during
PCR amplification. 5mC residues, however, are not converted and remain as cytosines after PCR amplification [93]. The development of high-throughput sequencing
has facilitated the generation of genome-wide, single-base resolution DNA methylation maps from bisulfite-converted DNA [94]. The first methylome was generated
in 2008 from the Arabidopsis thaliana genome [95, 96]. The first human methylome
was reported by Lister et al. in 2009 from human embryonic stem cells and fetal
fibroblasts [97]. These studies revealed the genome-wide context of DNA methylation. Extensive differences were found between the methylomes of two human cell
types, revealing a dynamic nature of this modification. Due to the high genomic
coverage of WGBS (95%), this method is preferred for building reference
methylomes [98].
WGBS can be combined with nucleosome occupancy and methylome sequencing
(NOMe-seq) to measure the relationship between DNA methylation and nucleosome
occupancy [99]. Developed by Jones et al. in 2012, this method utilizes the DNA
methyltransferase M.CviPI to methylate cytosines that are present in a GpC context.
GpC sites that are not protected by nucleosomes or transcription factors are accessible and can be methylated. M.CviPI does not alter endogenous DNA methylation
patterns found in a CpG context; thus, NOMe-seq can be paired with WGBS, and the
methylation status of both the CpG and GpC sites can be simultaneously detected
[100]. This method has enabled footprinting of chromatin architecture at a variety of
promoter and non-promoter regions. Work done by Jones et al. has demonstrated
that the relationship between nucleosome occupancy and DNA methylation is
context-specific, depending on genomic location [99]. Commercially available
NOMe-seq kits are available through Active Motif.
3.6 Reduced Representation Bisulfite Sequencing (RRBS)
Although WGBS allows for the greatest coverage of DNA methylation genomewide, it is time consuming and requires a significant investment to reach the effective
depth required for accurate DNA methylation mapping [101]. Although the cost of
WGBS has decreased significantly in recent years, it remains around $1,000 per
genome according to the National Human Genome Research Institute [102], making
this approach non-practical for large studies involving hundreds of samples
[103]. Reduced representation bisulfite sequencing (RRBS) is an efficient and
Experimental Methodologies for Detection and Mapping of Epigenetic DNA Marks
505
Studies of DNA methylation were initially restricted to localized CpG-rich regions
of the genome [92]. However, several methods now exist which can map DNA
methylation genome-wide. Whole-genome bisulfite sequencing (WGBS) was developed in 1992 by Frommer et al. to measure CpG methylation genome-wide at a
single-base resolution [93]. This method utilizes bisulfite-induced modification of
genomic DNA to convert cytosine to uracil; the latter is converted to thymine during
PCR amplification. 5mC residues, however, are not converted and remain as cytosines after PCR amplification [93]. The development of high-throughput sequencing
has facilitated the generation of genome-wide, single-base resolution DNA methylation maps from bisulfite-converted DNA [94]. The first methylome was generated
in 2008 from the Arabidopsis thaliana genome [95, 96]. The first human methylome
was reported by Lister et al. in 2009 from human embryonic stem cells and fetal
fibroblasts [97]. These studies revealed the genome-wide context of DNA methylation. Extensive differences were found between the methylomes of two human cell
types, revealing a dynamic nature of this modification. Due to the high genomic
coverage of WGBS (95%), this method is preferred for building reference
methylomes [98].
WGBS can be combined with nucleosome occupancy and methylome sequencing
(NOMe-seq) to measure the relationship between DNA methylation and nucleosome
occupancy [99]. Developed by Jones et al. in 2012, this method utilizes the DNA
methyltransferase M.CviPI to methylate cytosines that are present in a GpC context.
GpC sites that are not protected by nucleosomes or transcription factors are accessible and can be methylated. M.CviPI does not alter endogenous DNA methylation
patterns found in a CpG context; thus, NOMe-seq can be paired with WGBS, and the
methylation status of both the CpG and GpC sites can be simultaneously detected
[100]. This method has enabled footprinting of chromatin architecture at a variety of
promoter and non-promoter regions. Work done by Jones et al. has demonstrated
that the relationship between nucleosome occupancy and DNA methylation is
context-specific, depending on genomic location [99]. Commercially available
NOMe-seq kits are available through Active Motif.
3.6 Reduced Representation Bisulfite Sequencing (RRBS)
Although WGBS allows for the greatest coverage of DNA methylation genomewide, it is time consuming and requires a significant investment to reach the effective
depth required for accurate DNA methylation mapping [101]. Although the cost of
WGBS has decreased significantly in recent years, it remains around $1,000 per
genome according to the National Human Genome Research Institute [102], making
this approach non-practical for large studies involving hundreds of samples
[103]. Reduced representation bisulfite sequencing (RRBS) is an efficient and
Experimental Methodologies for Detection and Mapping of Epigenetic DNA Marks
505