high-throughput technique used to analyze methylation profiles at a singlenucleotide level from regions of high CpG content [104, 105]. This technique
utilizes a methylation-insensitive restriction enzyme, such as MspI, which cuts at
5
0 -CCGG-3
0 sites, enriching for CpG-rich regions of the genome such as CpG
islands by leaving CpG-rich regions at the ends of fragments being sequenced
[94]. This portion of the genome is then sequenced using NGS to generate singlebase pair resolution DNA methylation maps. RRBS captures 85% of CpG islands
and 60% of gene promoters, requiring small sample input (0.01–0.03 μg of DNA)
[94, 106]. By combining different restriction enzymes, CpG coverage across the
genome can be altered to include or exclude certain regions of interest such as CpG
island shores, which are the 2 kb regions adjacent to CpG islands and are known to
play an important role in various biological processes including cellular differentiation [107, 108].
One of the main advantages of RRBS over WGBS is the overall reduction of the
size of DNA regions to be sequenced, therefore requiring fewer reads for accurate
sequencing and thus reducing the cost and allowing for deep sequencing [94]. An
important disadvantage of RRBS is that it does not capture all CpG islands and/or
promoters. While WGBS can access ~95% of the genome, RRBS only reaches 3.7%
genome coverage [94]. However, RRBS enjoys much better coverage depth than
WGBS, and the concordance of DNA methylation levels between the two methods is
high [101]. Due to these advantages, RRBS has become popular when highthroughput, low-cost methylation analysis is needed such as in clinical applications
[109]. It is important to note, however, that RRBS has been mostly applied for DNA
methylation profiling of CpG sites within mouse and human genomes, and the ability
of MspI coupled with RRBS to cover non-CpG methylation has not been demonstrated [101]. MspI-based RRBS methods can only cover 10% of the genomic
regions [106].
More targeted capture-based approaches are available for sequencing specific
regions, such as the SeqCap Epi (Roche) and SureSelect (Agilent) systems
[110, 111]. These capture methods focus on predetermined genomic regions to
reduce sequencing complexity but are limited by design around specific genomic
regions which may or may not include the regions of interest. Custom capture
libraries can be created if certain regions are not part of the original capture
systems [112].
3.7 Modified Bisulfite Sequencing Methods for Detection
of 5hmC, 5fC, and 5caC
The discovery of 5hmC, 5fC, and 5caC as oxidation products of 5mC generated by
TET enzymes has spurred a flurry of speculation about possible biological roles of
these additional epigenetic marks [113]. In addition to their role as demethylation
intermediates, these oxidized forms of 5mC may serve as unique epigenetic marks in
506
C. L. Seiler et al.
utilizes a methylation-insensitive restriction enzyme, such as MspI, which cuts at
5
0 -CCGG-3
0 sites, enriching for CpG-rich regions of the genome such as CpG
islands by leaving CpG-rich regions at the ends of fragments being sequenced
[94]. This portion of the genome is then sequenced using NGS to generate singlebase pair resolution DNA methylation maps. RRBS captures 85% of CpG islands
and 60% of gene promoters, requiring small sample input (0.01–0.03 μg of DNA)
[94, 106]. By combining different restriction enzymes, CpG coverage across the
genome can be altered to include or exclude certain regions of interest such as CpG
island shores, which are the 2 kb regions adjacent to CpG islands and are known to
play an important role in various biological processes including cellular differentiation [107, 108].
One of the main advantages of RRBS over WGBS is the overall reduction of the
size of DNA regions to be sequenced, therefore requiring fewer reads for accurate
sequencing and thus reducing the cost and allowing for deep sequencing [94]. An
important disadvantage of RRBS is that it does not capture all CpG islands and/or
promoters. While WGBS can access ~95% of the genome, RRBS only reaches 3.7%
genome coverage [94]. However, RRBS enjoys much better coverage depth than
WGBS, and the concordance of DNA methylation levels between the two methods is
high [101]. Due to these advantages, RRBS has become popular when highthroughput, low-cost methylation analysis is needed such as in clinical applications
[109]. It is important to note, however, that RRBS has been mostly applied for DNA
methylation profiling of CpG sites within mouse and human genomes, and the ability
of MspI coupled with RRBS to cover non-CpG methylation has not been demonstrated [101]. MspI-based RRBS methods can only cover 10% of the genomic
regions [106].
More targeted capture-based approaches are available for sequencing specific
regions, such as the SeqCap Epi (Roche) and SureSelect (Agilent) systems
[110, 111]. These capture methods focus on predetermined genomic regions to
reduce sequencing complexity but are limited by design around specific genomic
regions which may or may not include the regions of interest. Custom capture
libraries can be created if certain regions are not part of the original capture
systems [112].
3.7 Modified Bisulfite Sequencing Methods for Detection
of 5hmC, 5fC, and 5caC
The discovery of 5hmC, 5fC, and 5caC as oxidation products of 5mC generated by
TET enzymes has spurred a flurry of speculation about possible biological roles of
these additional epigenetic marks [113]. In addition to their role as demethylation
intermediates, these oxidized forms of 5mC may serve as unique epigenetic marks in
506
C. L. Seiler et al.