bisulfite conversion which identifies cytosines as methylated or not methylated,
without distinguishing between 5mC and 5hmC. The other fraction undergoes
TAB conversion in which DNA is first glucosylated to protect 5hmC and then
oxidized by TET1, followed by bisulfite treatment. This allows 5hmC to be distinguished from 5mC [79].
3.3 Next-Generation Sequencing
Next-generation sequencing includes techniques that utilize polymerase synthesis of
complementary DNA strand to determine the sequence of input DNA. The technology, developed by Illumina (San Diego, CA), immobilizes DNA fragments onto a
chip by hybridizing them to synthetic DNA oligomers that were seeded on the chip
during manufacturing. The immobilized DNA is then amplified to create clusters of
identical DNA fragments, which will amplify the recorded signals. Following
amplification, the resulting DNA clusters are sequenced by synthesis of the complementary DNA using engineered DNA polymerases and specially modified
nucleic acids [80]. Each nucleotide has a fluorescent tag and is reversibly modified
at the 3
0 end to allow for only a single-nucleotide incorporation [80]. Following
single-nucleotide addition, a camera images the chip to record a fluorescent signal
and records the sequence being made, followed by deprotection of the 3
0 end for
continued synthesis [80]. These steps for elongation are repeated until the desired
read length is achieved. This technology can be combined with antibody/enrichment-based sequencing (Sect. 3.4), whole-genome sequencing (Sect. 3.5), reduced
representation sequencing (Sect. 3.6), and modified bisulfite sequencing (Sect. 3.7)
as detailed below.
3.4 Antibody/Enrichment-Based Sequencing
Low-resolution data for genome-wide distribution of DNA methylation marks can
be obtained using antibody-based techniques such as methylated DNA immunoprecipitation (MeDIP). In this assay, a 5mC-targeted antibody is used to specifically
immunocapture methylated genomic fragments [81]. The enriched DNA fragments
can then be input into high-throughput DNA detection methods such as DNA
microarray (MeDIP-Chip) or next-generation sequencing (MeDIP-Seq). Through
genome-wide peak calling of the microarray and sequencing results, methylated
regions along the whole genome can be identified [81, 82]. The advantages of this
method are its low cost and the ability to rapidly identify DNA methylation regions
within the genome. However, the enrichment efficiency of 5mC antibody can be
affected by the number of methylated CpG sites [81]. Thus, the detection sensitivity
is poor at low-CpG-density regions, making it hard to differentiate low methylation
differences in these regions.
Experimental Methodologies for Detection and Mapping of Epigenetic DNA Marks
503
without distinguishing between 5mC and 5hmC. The other fraction undergoes
TAB conversion in which DNA is first glucosylated to protect 5hmC and then
oxidized by TET1, followed by bisulfite treatment. This allows 5hmC to be distinguished from 5mC [79].
3.3 Next-Generation Sequencing
Next-generation sequencing includes techniques that utilize polymerase synthesis of
complementary DNA strand to determine the sequence of input DNA. The technology, developed by Illumina (San Diego, CA), immobilizes DNA fragments onto a
chip by hybridizing them to synthetic DNA oligomers that were seeded on the chip
during manufacturing. The immobilized DNA is then amplified to create clusters of
identical DNA fragments, which will amplify the recorded signals. Following
amplification, the resulting DNA clusters are sequenced by synthesis of the complementary DNA using engineered DNA polymerases and specially modified
nucleic acids [80]. Each nucleotide has a fluorescent tag and is reversibly modified
at the 3
0 end to allow for only a single-nucleotide incorporation [80]. Following
single-nucleotide addition, a camera images the chip to record a fluorescent signal
and records the sequence being made, followed by deprotection of the 3
0 end for
continued synthesis [80]. These steps for elongation are repeated until the desired
read length is achieved. This technology can be combined with antibody/enrichment-based sequencing (Sect. 3.4), whole-genome sequencing (Sect. 3.5), reduced
representation sequencing (Sect. 3.6), and modified bisulfite sequencing (Sect. 3.7)
as detailed below.
3.4 Antibody/Enrichment-Based Sequencing
Low-resolution data for genome-wide distribution of DNA methylation marks can
be obtained using antibody-based techniques such as methylated DNA immunoprecipitation (MeDIP). In this assay, a 5mC-targeted antibody is used to specifically
immunocapture methylated genomic fragments [81]. The enriched DNA fragments
can then be input into high-throughput DNA detection methods such as DNA
microarray (MeDIP-Chip) or next-generation sequencing (MeDIP-Seq). Through
genome-wide peak calling of the microarray and sequencing results, methylated
regions along the whole genome can be identified [81, 82]. The advantages of this
method are its low cost and the ability to rapidly identify DNA methylation regions
within the genome. However, the enrichment efficiency of 5mC antibody can be
affected by the number of methylated CpG sites [81]. Thus, the detection sensitivity
is poor at low-CpG-density regions, making it hard to differentiate low methylation
differences in these regions.
Experimental Methodologies for Detection and Mapping of Epigenetic DNA Marks
503