Download the manifest file for the array from the Product Files page of the BeadChip support pages.
For information, see the Infinium Methylation Coverage Technical Note.
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.
Similar to the Infinium genotyping assay, Illumina recommends DNA that is sized ≥ 2kb, which can be assessed on an agarose gel.
Yes. Illumina recommends the following of-the-shelf bisulfite conversion kits from Zymo Research.
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 MethylationEPIC Array using an FFPE Methylation Protocol along with the Infinium HD FFPE DNA Restore Kit.
To run FFPE samples on the InfiniumMethylationEPIC Array, perform the following procedures:
1. QC extract FFPE samples with the Infinium FFPE QC Kit.
2. Perform bisulfite conversion on only samples that pass QC with one of the approved kits.
3. Restore bisulfite-converted samples with the Infinium HD FFPE DNA Restore Kit.
4. Input restored FFPE samples into the Infinium Methylation Assay.
All DNA samples used with the Infinium HD Methylation assay, whether fresh/frozen or formalin-fixed, paraffin-embedded (FFPE) require a Zymo bisulfite conversion kit (with modified protocol). FFPE samples also require FFPE QC Assay and Infinium HD FFPE DNA Restore kits. For more information about the workflows for different sample types, see this bulletin.
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, therefore much of the DNA are denatured and more difficult to quantitate accurately.
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:
The scan takes ~18-37 minutes per BeadChip.
LIMS support is available for the Infinium MethylationEPIC BeadChip and the Infinium Mouse Methylation BeadChip.
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:
Samples from the same individual:
Samples from different persons:
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). For the Infinium HumanMethylation450K, Infinium Mouse Methylation, and Infinium MethylationEPIC array, we expect technical replicates to show > 98% correlation, and often observe > 99% correlation.