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Design, measurement and processing of region-specific DNA methylation assays: the mass spectrometry-based method EpiTYPER.

Suchiman HE, Slieker RC, Kremer D, Slagboom PE, Heijmans BT, Tobi EW - Front Genet (2015)

Bottom Line: The technology targets genomic regions of 100-600 base pairs and results in the quantitative measurement of DNA methylation levels largely at single-nucleotide resolution.It is particularly suitable for larger scale efforts to study candidate regions or to validate regions from genome-wide DNA methylation studies.Here, we describe in detail how to design and perform EpiTYPER measurements and preprocess the data, providing details for high quality measurements not provided in the standard EpiTYPER protocol.

View Article: PubMed Central - PubMed

Affiliation: Molecular Epidemiology, Department of Medical Statistics and Bioinformatics, Leiden University Medical Center Leiden, Netherlands.

ABSTRACT
EpiTYPER® is a mass spectrometry-based bisulfite sequencing method that enables region-specific DNA methylation analysis in a quantitative and high-throughput fashion. The technology targets genomic regions of 100-600 base pairs and results in the quantitative measurement of DNA methylation levels largely at single-nucleotide resolution. It is particularly suitable for larger scale efforts to study candidate regions or to validate regions from genome-wide DNA methylation studies. Here, we describe in detail how to design and perform EpiTYPER measurements and preprocess the data, providing details for high quality measurements not provided in the standard EpiTYPER protocol.

No MeSH data available.


Related in: MedlinePlus

Fragmentation of LEP promoter amplicon. The fragmentation of the LEP amplicon is given in gray lines. Red dots denote the measureable CpG unit. Clear from the figure is that four out of five CpG unit are measureable and that each CpG dinucleotide is contained in a separate fragment.
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Figure 4: Fragmentation of LEP promoter amplicon. The fragmentation of the LEP amplicon is given in gray lines. Red dots denote the measureable CpG unit. Clear from the figure is that four out of five CpG unit are measureable and that each CpG dinucleotide is contained in a separate fragment.

Mentions: First, we used the UCSC genome browser to gain the sequence of the LEP promoter, acquiring the first 5 kb upstream of the transcription start site and annotated frequent SNPs and repetitive elements. Using Epidesigner we designed multiple amplicons for the region including one spanning for 290 bases along chr7 and overlapping a CTCF binding site according to ENCODE CHIP-seq data (Figure 3). As CTCF binding may be methylation dependent, we took this amplicon further for characterization. This sequence contained no frequent SNPs that could interfere with the reliability of the measurements as shown in Table 3. The amplicon contains 5 CpG sites, of which one cannot be measured because the mass of the CpG unit is too low (Figure 4). The ampliconPredict function (RSeqMeth, 2008) indicates that none of the CpG units overlap in mass, have an identical mass, nor are any fragments located within 16 Da of other fragments (Table 4).


Design, measurement and processing of region-specific DNA methylation assays: the mass spectrometry-based method EpiTYPER.

Suchiman HE, Slieker RC, Kremer D, Slagboom PE, Heijmans BT, Tobi EW - Front Genet (2015)

Fragmentation of LEP promoter amplicon. The fragmentation of the LEP amplicon is given in gray lines. Red dots denote the measureable CpG unit. Clear from the figure is that four out of five CpG unit are measureable and that each CpG dinucleotide is contained in a separate fragment.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4585020&req=5

Figure 4: Fragmentation of LEP promoter amplicon. The fragmentation of the LEP amplicon is given in gray lines. Red dots denote the measureable CpG unit. Clear from the figure is that four out of five CpG unit are measureable and that each CpG dinucleotide is contained in a separate fragment.
Mentions: First, we used the UCSC genome browser to gain the sequence of the LEP promoter, acquiring the first 5 kb upstream of the transcription start site and annotated frequent SNPs and repetitive elements. Using Epidesigner we designed multiple amplicons for the region including one spanning for 290 bases along chr7 and overlapping a CTCF binding site according to ENCODE CHIP-seq data (Figure 3). As CTCF binding may be methylation dependent, we took this amplicon further for characterization. This sequence contained no frequent SNPs that could interfere with the reliability of the measurements as shown in Table 3. The amplicon contains 5 CpG sites, of which one cannot be measured because the mass of the CpG unit is too low (Figure 4). The ampliconPredict function (RSeqMeth, 2008) indicates that none of the CpG units overlap in mass, have an identical mass, nor are any fragments located within 16 Da of other fragments (Table 4).

Bottom Line: The technology targets genomic regions of 100-600 base pairs and results in the quantitative measurement of DNA methylation levels largely at single-nucleotide resolution.It is particularly suitable for larger scale efforts to study candidate regions or to validate regions from genome-wide DNA methylation studies.Here, we describe in detail how to design and perform EpiTYPER measurements and preprocess the data, providing details for high quality measurements not provided in the standard EpiTYPER protocol.

View Article: PubMed Central - PubMed

Affiliation: Molecular Epidemiology, Department of Medical Statistics and Bioinformatics, Leiden University Medical Center Leiden, Netherlands.

ABSTRACT
EpiTYPER® is a mass spectrometry-based bisulfite sequencing method that enables region-specific DNA methylation analysis in a quantitative and high-throughput fashion. The technology targets genomic regions of 100-600 base pairs and results in the quantitative measurement of DNA methylation levels largely at single-nucleotide resolution. It is particularly suitable for larger scale efforts to study candidate regions or to validate regions from genome-wide DNA methylation studies. Here, we describe in detail how to design and perform EpiTYPER measurements and preprocess the data, providing details for high quality measurements not provided in the standard EpiTYPER protocol.

No MeSH data available.


Related in: MedlinePlus