Quantify DNA Methylation with Predesigned PyroMark CpG Assays

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Minimize time spent on assay optimization and maximize analysis success
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PyroMark CpG Assays are the only genomewide, predesigned methylation assays for Pyrosequencing analysis that use a tailored design algorithm for highly specific assay design and successful CpG methylation results. Our upgraded assay database now includes over 84,000 individual assays for the human, mouse, and rat genomes. We also now offer predesigned assays for validating results from methylation arrays, such as the Infinium HumanMethylation450K and the MethylationEPIC BeadChip arrays.
Features and benefits
Highly successful PCR and Pyrosequencing
Assays for analyzing gene-specific CpG sites
Assays for validation of methylation array data
Features and benefits
PyroMark CpG Assays are predesigned DNA methylation assays for Pyrosequencing analysis, delivering highly specific quantification of CpG methylation for individual or multiple CpG sites. Each ready-to-use assay includes PCR and sequencing primers that have been designed by a rigorously tailored algorithm, along with an assay setup file and information about the CpG sites covered by the assay.

Benefits of PyroMark CpG Assays include:
  • Advanced assay design algorithms provide optimized results
  • Comprehensive coverage of CpG sites in the human, mouse, and rat genomes
  • Convenient download of run files and analysis settings
  • Easy-to-order predesigned assays for gene-specific or methylation array CpG sites
  • Top performance from a cost-effective solution

Highly successful PCR and Pyrosequencing
Successful methylation quantification by Pyrosequencing requires highly specific and unbiased PCR amplification. Bisulfite converted DNA is a less complex template and can be challenging to amplify specifically and at high yields. All primers in PyroMark CpG Assays have been checked against the entire human, mouse, or rat bisulfitome and are delivered at optimal concentrations for high performance. Together with the unique DNA protect technology of EpiTect Fast Bisulfite Conversion Kits and the carefully formulated reagents of the PyroMark PCR Kit, these predesigned assays ensure high yield and quality of sequencing template and increase reliability of downstream Pyrosequencing analyses.

Evaluation of PCR performance for 310 randomly selected PyroMark CpG Assays showed that 305 assays (98%) produced the predicted PCR amplicon, suitable for use as Pyrosequencing template. From these assays, 281 (92%) provided successful Pyrosequencing results (see figures High PCR success rate with bisulfite converted DNA and High Pyrosequencing success rate of predesigned PyroMark CpG Assays). These high-performing assays eliminate the need for tedious assay optimization and shorten the time to reliable results.
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Assays for analyzing gene-specific CpG sites
PyroMark CpG Assays enable methylation analysis of specific targets across the genome. Our database now contains over 84,000 predesigned assays covering more than 80% of gene-specific CpG islands for the human, mouse, and rat genomes. These assays are designed using a carefully tailored algorithm that generates optimal assay design based on bisulfite converted DNA. As such, these fast and easy-to-order assays minimize time spent on assay optimization and maximize analysis success. PyroMark CpG Assays expand the streamlined Pyrosequencing workflow to enable methylation analysis of virtually any CpG island in the human, mouse, or rat genome.
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Assays for methylation array validation
Hybridization arrays enable high-throughput discovery of variations in DNA methylation, but the results must be experimentally verified to confirm that the correct variations are identified. Pyrosequencing is a cost-effective technique highly suited for this purpose, providing quantitative analysis of short- to medium-length sequences with high accuracy. A recent article from Roessler and colleagues describes analysis of genomewide aberrations in DNA methylation using the Infinium HumanMethylation450K BeadChip array, followed by quantification of methylation levels for 692 individual CpG sites from 62 different genes using Pyrosequencing (1). The results of this cross-validation study showed very good concordance between both methods, confirming Pyrosequencing as a method highly suited for validation of array results.

QIAGEN now offers predesigned PyroMark CpG Assays for validating results from HumanMethylation450K and MethylationEPIC BeadChip experiments. These new predesigned assays for methylation array validation offer a convenient way to verify experimental results, and determine exact methylation levels of CpG sites of interest, without the need to design a separate assay for each individual target of interest. Our dedicated search page lets users simply enter the CpG loci identification numbers (CG#) of interest to easily search our assay database and order the ready-to-use PyroMark CpG Assays. If an assay is not already available for a particular target, one will be automatically designed within just a few minutes using our dedicated algorithm and made available in our database.
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References
1. Roessler, J., Ammerpohl, O., Gutwein, J., Hasemeier, B., Anwar, S.L., Kreipe, H., and Lehmann, U. (2012) Quantitative cross-validation and content analysis of the 450k DNA methylation array from Illumina, Inc. BMC Research Notes. 5:210.

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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.
Improved methylation quantification in homopolymers
Improved 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.
Pyrosequencing analysis of CpG methylation pattern in the RASSF1A gene
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
Linearity of methylation quantification.
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.
High PCR success rate, even with bisulfite-converted DNA.
High PCR success rate, even with bisulfite converted DNA.
The PCR performance of PyroMark CpG Assays for 310 randomly selected genes was evaluated. A total of 305 assays (98%) resulted in the predicted PCR amplicon, usable as template for subsequent Pyrosequencing.
High Pyrosequencing success rate of predesigned PyroMark CpG Assays
High Pyrosequencing success rate of predesigned PyroMark CpG Assays.
The Pyrosequencing performance of PyroMark CpG Assays for 310 randomly selected genes was evaluated. A total of 305 assays (98%) resulted in the predicted PCR amplicon, usable as template for subsequent Pyrosequencing. From these assays, 281 resulted in successful Pyrosequencing results. Of these successful assays, 229 (75.1%) required no further review by the user, and 52 (17%) were successful after user review. Only 24 (7.9%) evaluated assays failed.
Quantification of CpN methylation
Quantification of 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.