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 off-the-shelf bisulfite conversion kits from Zymo Research.
Lightning Kits from Zymo Research are offered in manual and automated 1-hour bisulfite conversion reaction kits:
Where minimal deviation from historic studies with EPIC, 450K, and 27K is of primary importance, Zymo Research’s Classic EZ DNA Methylation kits (1.5 day manual workflow) are recommended:
FFPE samples are not supported currently.
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 is denatured and more difficult to quantitate accurately.
Illumina LIMS is not supported at this time.
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.
The GenomeStudio Methylation Module extracts beta values and provides sample clustering analysis for Infinium HTS iSelect Methyl Custom BeadChip data. The BeadArray Controls Reporter software tool is not supported at this time.
Notably, there are many freeware applications in Bioconductor, such as SeSAMe, which provide enhanced normalization and visualization options. Illumina does not support these freeware solutions directly.
The standard GenomeStudio and SNP controls that are available on the EPIC BeadChip are included.
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).