Infinium MethylationEPIC BeadChip FAQs

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  • Analysis

  • The GenomeStudio Methylation Module extracts beta values and provides clustering and normalization for Infinium MethylationEPIC array data. In addition, there are many freeware applications in Bioconductor, such as chAMP and RnBeads, which provide enhanced normalization and visualization options. Illumina does not support these freeware solutions directly.

    SNPs were included on the BeadChip so investigators could generate a DNA fingerprintof their samples as an added level of quality control. Please find further information in the Infinium HD Methylation Assay Protocol Guide. SNP assays on the BeadChip are not mentioned in the assay guide and only briefly described in the GenomeStudio Methylation Module. Follow this method to confirm the identity of samples from the same individual:

    1. Highlight the SNP assays in the sample methylation profile, right-click, and select Show only selected rows.
    2. For any given pair of samples that are supposed to be from the same individual, plot the beta values in a scatter plot from the sample methylation profile.

      Samples from the same individual:

      Infinium HumanMethylation450 samples from one individual.

      Samples from different persons:

      Infinium HumanMethylation450 samples from different people.

      The scatterplots differ because the beta values calculated from SNP assays will cluster, much as they do in a standard genotyping theta graph. Samples from the same individual have the SNP results fall along the identity line in a scatter plot, whereas samples from different persons scatter into the 9 different possible spots, based on their genotypes.

    Background subtraction is required when comparing data collected with different scanners or processing attributors (eg, date, reagent lot, instrument, operator, etc.). Background subtraction has a much smaller effect when you scan chips on the same scanner, and might not be necessary. Analyze a subset of data with and without subtraction, and choose the subset of data you prefer based on your results.

    What you are seeing are histograms of the beta values in bins of 0.02 steps and categorized by Infinium design type. The difference in beta value ranges between the Infinium I and Infinium II assay design types cause the double peaks. In general, the beta peaks at the extremes of Infinium I probes tend to be further out than the beta peaks for Infinium II.

    The beta beak differences do not affect the final analysis of the project. Individual CpG assays are not intended to be compared directly with other CpG assays, as each probe (or probe set for Infinium I designs) has different binding characteristics and behaves differently than any other probe or probe set. Rather, each assay is compared between two samples or sample groups (ie, in determining a relative rather than an absolute methylation value).