Questions & Answers

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Questions & Answers

  • Is there LIMS support for Infinium Methylation BeadChips?

    LIMS support is available for the Infinium HumanMethyation450 BeadChip and Infinium MethylationEPIC BeadChip.

  • Does Illumina recommend background subtraction for analysis?

    Background subtraction is required when you compare data run on different types of scanners because of technical disparities; for example, the iScan and HiScan have different offsets. 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.

  • For the bisulfite conversion, do I need specific hardware?

    Perform this reaction in a thermal cycler to keep the temperature stable. Any PCR machine can be used for the bisulfite conversion reactions, but this reaction must be performed in the pre-PCR room.

  • Can I use the modified cycling incubation protocol for the bisulfite conversion recommended for Infinium Methylation analysis on samples to be analyzed on the GoldenGate Methylation protocol?

    Yes, the modified cycling incubation can be used on the GoldenGate Methylation Assay. It is not required, however, because that assay is more tolerant towards incomplete conversion.

  • How much DNA is required for the Infinium Methylation Assay?

    Going into the bisulfite conversion, at least 250 ng is required for the manual protocol, and at least 1000 ng is required for the automated protocol. Using less than the recommended amount does not guarantee R2 performance to meet specifications.

  • Why doesn't the Infinium HumanMethylation450 BeadChip contain specific distances from the CpG site to the transcriptional start site (TSS) of the listed genes?

    The array density is much higher in the Infinium HumanMethylation450 BeadChip. There are a large number of CpGs for which multiple transcripts are listed and for which the CpG site can fall into different annotation categories. Consequently, the task of calculating TSS distances would be a large bioinformatic undertaking, and the numbers would have to be modified every time the genome was updated. Additionally, the column "UCSC_RefGene_Group" has content about the location of the CpG relative to specific regions and features of the associated genes, which is in many ways richer than the simple distance relative to transcriptional start site.

  • Can I use the Zymo EZ DNA Gold kit for bisulfite conversion?

    Yes, you can, but you must use this kit for your entire project. That means you cannot use a mixture of samples that have been converted with different kits on the same arrays, or within the same project.

  • Can I run FFPE samples on the Infinium Methylation Assay?

    FFPE samples are NOT recommended for the standard protocol. FFPE samples are already highly degraded, with a high level of crosslinking, so conversion does not occur effectively. However, you can run FFPE samples on the Infinium HumanMethylation450 BeadChip using the FFPE automated or FFPE manual protocol along with the Infinium FFPE DNA Restoration Solution kit.

  • Is the controls dashboard different for the Infinium HumanMethylation27 BeadChip versus the HumanMethylation450 BeadChip?

    Yes, there are several differences in the control dashboard due to the inclusion of Infinium II Assay designs for the Infinium HumanMethylation450 BeadChip. Please see the Infinium HumanMethylation450 BeadChip User Guide for more details.

  • Does Illumina recommend any the commercially available methylation standard DNA: methylated, hemimethylated, or unmethylated?

    Illumina has not extensively tested any of the commercially available standards, and does not currently recommend any of them. In-house, Illumina uses standards generated in our own laboratory. Our methods for generating the standards are described in the following article:

    Bibikova M, Le J, Barnes B, Saedinia-Melnyk S, Zhou L, et al. (2009) Genome-wide DNA methylation profiling using Infinium assay, Epigenomics 1:177-200

  • Is the Infinium Methylation Assay a one-color or two-color assay?

    This is a two-color assay.

    For the Infinium HumanMethyation27 BeadChip assay, which is based on Infinium I Assay designs, the color incorporated depends upon the base preceding the CpG locus being queried. This can be either green or red.

    The Infinium HumanMethylation450 BeadChip assay includes Infinium I and Infinium II study designs.  In the latter case, a single base extension from the 3' end of the probe sequence (which is one base upstream of the query base) will result in either a red or green signal depending on whether the query site was unmethylated or methylated.

  • Does Illumina recommend specific bisulfite conversion kits?

    Yes. Illumina recommends the following of-the-shelf bisulfite conversion kits from Zymo Research.

    Catalog Number

    D5001    50 reactions (single-column format)

     

    D5002    200 reactions (single-column format)

     

    D5004    2×96 reactions EZ-96 DNA methylation kit (deep-well format)

  • Is the Infinium MethylationEPIC BeadChip controls dashboard similar to the Infinium HumanMethylation450 BeadChip?

    Yes, the same controls types are present.

  • Why are SNPs included in the manifest of the methylation BeadChip?

    SNPs were included on the BeadChip so investigators could generate a DNA "fingerprint" of 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 "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 is 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.

  • I have noticed double peaks in methylation value that seem to be related to Infinium I versus Infinium II probe designs. What is causing these double peaks?

    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.

    These 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 2 samples or sample groups (ie, in determining a relative rather than an absolute methylation value). For the Infinium HumanMethylation450 and Infinium MethylationEPIC array, we expect technical replicates to show > 98% correlation, and often observe > 99% correlation.

  • What size DNA is recommended for the Infinium Methylation Assay?

    Similar to the Infinium genotyping assay, we recommend DNA that is sized ≥ 2kb, which can be assessed on an agarose gel.

  • How are methylation levels measured by the BeadChip?

    A beta value (β) is calculated for each CpG locus.

  • Is requantification of DNA samples after bisulfite conversion recommended?

    If you use at least 250-1000 ng DNA for the bisulfite conversion, requantification is not necessary. It is critical to quantify the input DNA concentration with PicoGreen to make sure that you add sufficient DNA to the bisulfite conversion reaction. Bisulfite conversion renders DNA less complementary, so much of the DNA are denatured and more difficult to quantitate accurately.

  • Where can I find a marker list of all the CpG sites on the array?

    The manifest for the array can be found in the Downloads section of the BeadChip support page.

  • Where can I find information about integrating probes on a methylation BeadChip with probes on the HumanHT-12 Expression BeadChip?
  • Why are both Infinium I and Infinium II probes used for this BeadChip? Does the design affect the data output / quality?

    For more information, see the Infinium Methylation Coverage Technical Note.

  • Can the Infinium HumanMethylation450K BeadChip or the Infinium MethylationEPIC BeadChip distinguish 5-hydroxymethylcytosine from 5-methylcytosine?

    Illumina has not validated the array for 5-hMc. However, publications have used the Infinium HumanMethylation450K array for 5-hMc analysis and it is possible that this protocol will works on the Infinium MethylationEPIC array.

    For more information, see Nazor, Kristopher L., et al. "Application of a low cost array-based technique—TAB-Array—for quantifying and mapping both 5mC and 5hmC at single base resolution in human pluripotent stem cells." Genomics 104.5 (2014): 358-367.

  • How is the beta value calculated?

    The beta value (β) uses the ratio of intensities between methylated and unmethylated alleles to estimate the methylation level of the CpG locus.

  • What DNA quantification method does Illumina recommend for Infinium arrays?

    For Infinium arrays, Illumina recommends use of PicoGreen reagent for DNA quantification. UV-based methods may can overestimate DNA concentration by 2–10 fold.

  • Is there a GenTrain and/or cluster file for Infinium Methylation Arrays?

    Since Infinium Methylation arrays are designed to compare relative methylation levels between two samples or sample groups (such as normal versus tumor, or pancreas cells versus liver cells), there is no GenTrain and/or cluster file for this product. It is similar to doing a paired-sample analysis.