DNA Methylation Analysis

Pyrosequencing in epigenetics research

The study of epigenetic mechanisms has become increasingly prevalent in disciplines ranging from cancer research to genetic imprinting and biomarker development. Pyrosequencing provides accurate and detailed profiles of DNA methylation patterns underlying cell cycle regulation, differential gene expression, and epigenetic effects. Now, the recently launched PyroMark Q24 Advanced system makes it easy to quantify methylation at non-CpG (CpN) sites commonly found in embryonic stem cells and plants, expanding the application range of this powerful technique.

Benefits of Pyrosequencing

Pyrosequencing facilitates accurate DNA methylation analysis by:

Quantifying methylation in explicit sequence context
Measuring methylation status of individual and multiple CpG or non-CpG (CpN) sites
Permitting rapid, easy, and high-throughput analysis of methylation
Incorporating internal controls to confirm complete bisulfite treatment

Precise and quantitative methylation analysis

Standard methylation analysis methods provide only qualitative or semi-quantitative data, which can lead to inaccurate conclusions regarding the effects of epigenetic DNA methylation on cell cycle and metabolism. Without quantification of methylation levels, it is not possible to distinguish physiologically relevant methylation from background methylation.

Pyrosequencing solves this limitation by generating highly reproducible quantification of methylation frequencies at individual consecutive CpG sites (see figures Pyrosequencing analysis of CpG methylation pattern in the RASSF1A gene and Linearity of methylation quantification by Pyrosequencing). As such, Pyrosequencing can detect and quantify even small changes in methylation levels. Other valuable features include the inherent quality control afforded by the sequence context of results and the ability to compare results to expected methylation levels. Built-in controls for the bisulfite treatment eliminate manual estimation of non-converted DNA levels and prevent false-positive methylation detection, thereby ensuring the reliability of results.

Pyrosequencing analysis of CpG methylation pattern in the RASSF1A gene. The figure illustrates the variation in methylation level between 5 different CpG sites in 4 individual tumor samples. Each sample was run in duplicate, and the concordance between the two samples clearly illustrates the reproducibility of Pyrosequencing technology.

Linearity of methylation quantification by Pyrosequencing. Linearity of methylation quantification by Pyrosequencing. PCR products from varying mixtures of unmethylated genomic DNA and methylated DNA (EpiTect Control DNAs) were analyzed by Pyrosequencing. A tight correlation between the known percentage of methylated DNA in the mixtures (squares) and the methylation percentage reported by Pyrosequencing (triangles) was observed (r2 = 0.9962). The graph represents the quantification of methylation at a single CpG site in the p16 gene.

These features have established Pyrosequencing as the gold standard for DNA methylation analysis. This technology has been used to correlate DNA methylation to tumor type and gene expression, to measure cellular response to treatment with demethylating agents, and to assess changes in methylation state in relation to tumorigenesis, genetic imprinting, and exposure to environmental toxins (1).

Valuable quality control of bisulfite conversion

To characterize the methylation status of a DNA sequence via Pyrosequencing, the DNA is first incubated with sodium bisulfite. As a result, unmethylated cytosine residues are converted into uracil while methylated cytosines remain unchanged, giving rise to two different sequences that can be distinguished. An internal control for bisulfite treatment is incorporated into analysis. Cytosines that are not followed by guanine in template sequences are not methylated, and should therefore be converted to thymine by bisulfite treatment and PCR (see table Sequences resulting from bisulfite conversion and PCR). Full bisulfite conversion is confirmed if all templates show thymine and no cytosine in these positions. With unique DNA Protect technology, QIAGEN EpiTect Bisulfite Kits facilitate complete conversion and minimal degradation of the treated DNA.

Sequences resulting from bisulfite conversion and PCR

	Original sequence	After bisulfite treatment	After PCR amplification
Unmethylated DNA	A-C-G-T-C-G-T-C-A	A-U-G-T-U-G-T-U-A	A-T-G-T-T-G-T-T-A
Methylated DNA	A-C-G-T-C-G-T-C-A	A-C-G-T-C-G-T-U-A	A-C-G-T-C-G-T-T-A

Flexible analysis of methylation patterns

The analysis of methylation status exploits the quantitative nature of Pyrosequencing data. Unlike Sanger sequencing, the peak heights in the resulting Pyrogram report the ratio of cytosine to thymine at each analyzed CpG site, which reflects the proportion of methylated DNA. Assay design is flexible — it can be performed in forward or reverse orientations, on either the top or the bottom strands. In addition, contiguous CpG sites are analyzed independently and within the same run, which enables assessment of sequence-wide methylation patterns while retaining details of position specific methylation (see figure Analysis of 16 CpG sites in a long sequence run).

Analysis of 16 CpG sites in a long sequence run. PyroMark Q24 Advanced increases both read length and reliability of methylation analysis at positions later in the sequence. This example demonstrates 135 nucleotide dispensations and the accurate analysis of 16 different CpG positions in a single PyroMark Q24 Advanced CpG reaction. To accurately analyze all of these sites with PyroMark Q24, one would need to run 3 separate assays.

Quantification of methylation at non-CpG (CpN) sites

The new CpN mode of the PyroMark Q24 Advanced system now enables methylation analysis of cytosine residues that are not part of CpG sites. Analysis of CpN and CpG sites can be performed together in a single Pyrosequencing reaction. Each of these positions can be selected individually in the software during the run setup. This example shows the analysis of a sequence with a CpN site (CpA in this case) in the first position, followed by 2 classical CpG sites (see figure New mode for analyzing CpN methylation)

New mode for analyzing CpN methylation. The new CpN mode of Pyromark Q24 Advanced enables methylation analysis of cytosine residues that are not part of CpG sites. Each of these positions can be selected individually. This example shows the analysis of a CpN site (CpA in this case) in the first position, followed by 2 classical CpG sites.

Quantification of methylation at any sequence position

Previously, analysis of methylation sites further away from the sequencing primer could be uncertain, but now with longer read lengths and higher accuracy of the PyroMark Q24 Advanced system, methylation quantification is highly reliable throughout the entire sequencing run. Bisulfite conversion in DNA methylation analysis leads to frequent poly T stretches in the nucleotide sequence, and analysis of CpG sites directly after such T homopolymers has previously been challenging due to uncertain quantification of the light signal at these sites. The increased accuracy of PyroMark Q24 Advanced enables reliable quantification of CpG methylation behind and even within a stretch of 8 T nucleotides (see figure Methylation quantification in homopolymers).

Methylation quantification in homopolymers. Quantification of CpG methylation directly following a T homopolymer or between T homopolymers is especially challenging. Methylation levels are determined by the ratio of converted C nucleotides to unconverted C nucleotides following bisulfite treatment, and since the C to T conversion often leads to long T homopolymer stretches in an amplicon, this scenario occurs often. PyroMark Q24 Advanced enables accurate quantification of CpG position after or between T homopolymers.This example shows the analysis of a CpG site within a stretch of 8 T nucleotides.

References

Dejeux, E., El abdalaoui, H., Gut, I.G., and Tost, J. (2009) Identification and quantification of differentially methylated loci by Pyrosequencing Technology. Methods Mol. Biol. 507, 189.