5hmC significantly enhances its kinetics signature, making simultaneous mapping of
all three oxidized forms of 5mC possible [126]. Because these methods do not rely
on DNA conversion with bisulfite or enzymatic processes and allow for longer reads,
read alignment is simplified. Despite these positives, third-generation sequencing
still has areas which need improvement, specifically signal intensity for some
specific epigenetic marks, the overall accuracy, and larger DNA input requirements.
These limitations prevent the application of this technology to whole-genome
sequencing without some form of enrichment [29].
4.2 Nanopore Sequencing
Another emerging third-generation sequencing technology is nanopore sequencing,
which can directly read different bases out using distinct ionic current signals
generated as DNA translocates through a protein nanopore [127]. Generally, a
protein nanopore is set in an electrically resistant polymer membrane, and an ionic
current is passed through the nanopore by setting a voltage across this membrane.
When DNA passes through the pore or near its aperture, this event creates a
characteristic disruption in the current. Measurement of that current makes it possible to identify the molecule in question. Previous studies have shown the ability of
discriminating between all five cytosine variants by low-throughput nanopore sensors [128]. However, high-throughput nanopore sequencing can only discriminate
cytosine, 5mC, and 5hmC [129]. Notably, the overall error rate for nanopore
sequencing is 13.4–20.2%, which is higher as compared to SMRT sequencing
(1.72–14.2%) [130]. However, both third-generation sequencing techniques remain
under active development, which will hopefully bring more robust, cost-effective,
and straightforward solutions to the sequencing of epigenetic modifications in DNA.
5 Conclusions
A growing number of DNA epigenetic modifications identified in human cells
suggest that epigenetic DNA marks are more common in higher eukaryotes than
previously thought. DNA methylation and other epigenetic marks play a central role
in controlling the physiological processes of living cells. Epigenetics has been
shown to have broad implications for human health, which creates a need for reliable
techniques to detect and measure these epigenetic modifications. A variety of
methods have been developed and applied to study these epigenetic marks
(Table 2). Recent years have seen a marked increase in novel approaches to analyze
the expanding number of epigenetic DNA modifications. Information obtained from
such studies will be useful both for our understanding of basic cellular regulation and
for identifying future targets for drug design.
512
C. L. Seiler et al.
all three oxidized forms of 5mC possible [126]. Because these methods do not rely
on DNA conversion with bisulfite or enzymatic processes and allow for longer reads,
read alignment is simplified. Despite these positives, third-generation sequencing
still has areas which need improvement, specifically signal intensity for some
specific epigenetic marks, the overall accuracy, and larger DNA input requirements.
These limitations prevent the application of this technology to whole-genome
sequencing without some form of enrichment [29].
4.2 Nanopore Sequencing
Another emerging third-generation sequencing technology is nanopore sequencing,
which can directly read different bases out using distinct ionic current signals
generated as DNA translocates through a protein nanopore [127]. Generally, a
protein nanopore is set in an electrically resistant polymer membrane, and an ionic
current is passed through the nanopore by setting a voltage across this membrane.
When DNA passes through the pore or near its aperture, this event creates a
characteristic disruption in the current. Measurement of that current makes it possible to identify the molecule in question. Previous studies have shown the ability of
discriminating between all five cytosine variants by low-throughput nanopore sensors [128]. However, high-throughput nanopore sequencing can only discriminate
cytosine, 5mC, and 5hmC [129]. Notably, the overall error rate for nanopore
sequencing is 13.4–20.2%, which is higher as compared to SMRT sequencing
(1.72–14.2%) [130]. However, both third-generation sequencing techniques remain
under active development, which will hopefully bring more robust, cost-effective,
and straightforward solutions to the sequencing of epigenetic modifications in DNA.
5 Conclusions
A growing number of DNA epigenetic modifications identified in human cells
suggest that epigenetic DNA marks are more common in higher eukaryotes than
previously thought. DNA methylation and other epigenetic marks play a central role
in controlling the physiological processes of living cells. Epigenetics has been
shown to have broad implications for human health, which creates a need for reliable
techniques to detect and measure these epigenetic modifications. A variety of
methods have been developed and applied to study these epigenetic marks
(Table 2). Recent years have seen a marked increase in novel approaches to analyze
the expanding number of epigenetic DNA modifications. Information obtained from
such studies will be useful both for our understanding of basic cellular regulation and
for identifying future targets for drug design.
512
C. L. Seiler et al.