Quantitative analysis of methylation at multiple CpG sites by PyrosequencingTM

Robert England and Monica Pettersson Pyrosequencing from Biotage1 improves virtually every aspect of CpG methylation analysis: quantification, resolution, accuracy, built-in quality control, speed and ease of use. By reporting the individual degrees of methylation in up to 10 consecutive CpG sites in a single assay, Pyrosequencing gives a new dimension to CpG methylation analysis, thus opening the opportunity to reveal previously unseen patterns of methylation.

Methylation of cytosines in CpG dinucleotides is an important regulator of gene expression in the human genome. Changes in CpG methylation play a fundamental role in the development of a variety of tumours. Studies of CpG methylation have opened new opportunities for understanding cancer development and progression.

Pyrosequencing is ideal for the analysis of CpG methylation since it is uniquely capable of quantifying methylation in explicit sequence context, thereby enabling several consecutive CpG sites to be quantified individually in a single assay. The method is easy and fast to perform, is automated and relatively inexpensive, and offers higher processing throughput compared to other technologies.

Pyrosequencing assays are relatively easy to design, since there are relatively few constraints to the positioning of the sequencing primer. It can often be positioned in a region free of CpG sites, with flexibility for varying the distance from the first base to be sequenced. Furthermore, the assay gives the same quantitative measurement of CpG methylation when performed on either the top or the bottom strand, or in either the forward or reverse directions. There are therefore four possible assay designs for any given set of CpG sites to be analysed.

The application of the technology for CpG methylation analysis has been pioneered and verified in a number of independent studies (Colella et al, Tost et al, Uhlmann et al and Dupont et al). Yang et al extended the concept to provide estimates of global methylation from repetitive DNA elements.

1 Biotage AB, Kungsgatan 76, SE-753 18 Uppsala, Sweden Correspondence should be addressed to CpG@pyrosequencing.com

Principle of Analysis As with most methods for quantitative analysis of CpG methylation, CpG sites of genomic DNA are first chemically converted by bisulfite treatment and subsequently amplified by PCR. In the first step of this process, genomic DNA is treated with bisulfite, so that unmethylated Cytosine (C) is converted to Uracil (U), whereas methylated Cytosine (mC) remains unchanged. The second step is PCR amplification, where Uracil (U) is amplified to Thymine (T), and methylated Cytosine (mC) is amplified to Cytosine (C). Discrimination between mC and C is thereby achieved by transforming mC and C to appear as an artificial C/T SNP. The measured level of C is equal to the degree of methylation at each CpG site (Figure 1).

Figure 1. An example of a methylated DNA sequence and its conversion by bisulfite treatment and further amplification by PCR. By Pyrosequencing, unmethylated Cytosine, C is measured as the relative content of T at the CpG site, and methylated Cytosine, mC, is measured as the relative content of C at the CpG site.

Pyrosequencing Pyrosequencing accurately and reproducibly quantifies the ratio C/T (the artificial SNPs obtained from the CpG sites) by sequential addition of nucleotides to the single stranded DNA template (Figure 2). Briefly, Pyrosequencing employs an enzyme cascade system, consisting of 4 enzymes and specific substrates, to produce light whenever a nucleotide forms a base pair with the complementary base in the DNA template strand. The intensity of light is proportional to the number of nucleotide molecules incorporated (for a full description of Pyrosequencing, see Ronaghi et al).

Figure 2. The Pyrosequencing reaction cascade generates light for every incorporated nucleotide, the intensity of which is proportional to the number of bases incorporated.

Sequencing through the DNA template generates a PyrogramTM, a pictorial representation of sequential nucleotide dispensations plotted against the resultant measured light intensity (Figure 2). The Pyrogram displays both the nucleotide sequence (as peak sequence) as well as a quantitative representation of the incorporation events (as peak heights). The degree of methylation is calculated from the peak heights of the sequential C and T incorporations.

Built-in Quality Controls For the CpG methylation analysis method to be quantitative, all sources of variance during the chemical transformation and subsequent PCR amplification must be identified and minimised. The data generated by Pyrosequencing contain unique features that act as quality controls. First, the results of the analysis are presented in sequence context, confirming that the analysis was made at the correct sites. This is an important control for CpG methylation analysis, since bisulfite-treated, PCR-amplified DNA is very AT-rich and therefore has a low level of sequence variation. Second, when the assay includes analysis of a C not followed by a G, that C should be fully converted to T (blue column in Figure 1). This acts as a useful quality control for the full conversion of unmethylated C to T by the bisulfite treatment and subsequent PCR reaction.

Workflow Commercial reagent kits are available for bisulfite treatment of human genomic DNA. The PCR reaction is performed with one biotinylated PCR primer, which enables the conversion of the PCR product to a single-stranded DNA template suitable for Pyrosequencing. A sequencing primer is then added, which anneals to the single stranded DNA template. The preparation and analysis times given in Table 1 are for up to 96 samples run in parallel.

Step Time

Bisulfite treatment of genomic DNA (C → U, mC → mC) 3 hours hands-on, overnight incubation

PCR amplification (U → T, mC → C) 2 hours

Preparation of DNA templates for Pyrosequencing 5 minutes hands-on, 15 minutes total

Analysis of CpG methylation by Pyrosequencing 1 minute/dispensation

Calculation on C:T peak heights to give % methylation Approx. 1 minute

Table 1. Workflow of CpG methylation analysis by Pyrosequencing for up to 96 samples in parallel

Assay Results Pyrosequencing is capable of measuring degree of CpG methylation with high accuracy. Replicates usually vary by less than ±3% (Table 2). Assays are sufficiently sensitive to detect less than 10% methylation.

CpG site Pos 1 Pos 2 Pos 3 Pos 4

Mean value of 6 replicates % 38.1 37.2 35.5 35.6

SD % 1.2 2.1 0.9 1.2

Table 2. Mean CpG methylation of 4 consecutive CpG sites (labelled Pos 1-4) and standard deviations in 6 PCR replicates for a typical CpG methylation assay. The PCR reactions were run separately of each other, followed by Pyrosequencing analysis. Therefore the measured variance comes from the PCR reaction plus the Pyrosequencing analysis.

Results of a CpG methylation analysis are presented in Figure 3. This particular assay measures the degrees of methylation at 9 CpG sites over 82 bases.

Figure 3. A Pyrogram showing methylation in 9 consecutive CpG sites across 82 nucleotides. The orange columns highlight the peaks resulting from sequential dispensations of C and T from which methylation is assessed. Courtesy of Dr. Jörg Tost, Centre National de Génotypage, Evry, France.

Conclusion CpG methylation is a rapidly growing area of research since its relevance in tumorigenesis is now established, apparent as both global genomic hypomethylation as well as epigenetic silencing through hypermethylation of CpG islands upstream of genes. Pyrosequencing clearly contributes to research knowledge by providing reliable and detailed data over multiple CpG sites to provide a sound basis for comparing methylation profiles over time and among individuals.

References Colella S, Shen L, Baggerly KA, Issa J-PJ, Krahe R. Sensitive and quantitative universal Pyrosequencing methylation analysis of CpG sites. BioTechniques, Jul 2003; 35: 146-150. Dupont JM, Tost J, Jammes H, and Gut IG. De novo quantitative bisulfite sequencing using the pyrosequencing technology. Anal Biochem, Oct 2004; 333(1): 119-27. Ronaghi, M. Pyrosequencing sheds light on DNA sequencing, Genome Research, Jan 2001; 11(1): 3-11. Tost J, Dunker J, Gut IG. Analysis and quantification of multiple methylation variable positions in CpG islands by PyrosequencingTM. BioTechniques. Jul 2003; 35: 152-156. Uhlmann K, Brinckmann A, Toliat MR, Ritter H, Nürnberg P. Evaluation of a potential epigenetic biomarker by quantitative methyl-single nucleotide polymorphism analysis. Electrophoresis. 2002; 23: 4072-4079. Yang AS, Estecio MR, Doshi K, Kondo Y, Tajara EH, Issa JP. A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements. Nucleic Acids Res. Feb 2004; 32(3): e38. BIOTAGE HEADQUARTERS US OFFICE EUROPEAN OFFICES

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