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.
Using the standard Infinium Methylation protocol, ≥ 96% of sites on the array should be detected by GenomeStudio, assuming high quality intact genomic DNA at a minimum of 250ng input per sample.
Illumina has not validated the array for 5-hMc. However, publications have used the Infinium HumanMethylation450K and MethylationEPIC BeadChips for 5-hMc analysis and it is possible that this protocol will works on the Infinium Mouse Methylation BeadChip.
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. While we have not tested our Infinium HD FFPE DNA Restore Kit with Mouse FFPE samples and do not guarantee the performance of this kit, you may attempt to use this kit for FFPE-derived mouse DNA samples. We do not offer an FFPE QC kit for mouse-derived DNA at this point in time.
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 has carried out a reasonable level of testing with commercial mouse DNA methylation standards from EpigenDX. The available DNA has increasing amounts of genome-wide methylation, and can be used to check the performance of the Infinium assay in resolving DNA methylation differences.
LIMS support is available for the Infinium Mouse Methylation BeadChip. For the specific Illumina LIMS module and version required for running the Mouse Methylation BeadChip, see Compatible Products.
The GenomeStudio Methylation Module extracts beta values and provides clustering for Infinium Mouse Methylation BeadChip data. GenomeStudio controls normalization with the Mouse Methylation BeadChip is not supported at this time. Also, 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.
SNPs were included on the BeadChip so investigators could generate a DNA fingerprint of their samples as an added level of quality control. Specifically, for the Mouse Methylation BeadChip, SNP probes are included to help identify the strain origin of the DNA being analyzed. 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.
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).
Specific studies, like work with patient-derived xenograft samples, usually result in mixtures of human and mouse DNA. We included human and mouse controls so that researchers could assess the performance integrity of DNA from both species on the same BeadChip. Human control probes have an addendum to their name, ie, HSA, while mouse control probes have a similar addendum noted as MUS.
Our technical support teams and customers have noted that samples showing abnormal data in these control probes almost always show abnormal behavior in other sample independent control probes. As these controls add no additional information, Illumina decided to remove these probes.
The IlmnID is a composite of multiple information fields: the name of the probe (locus target identifier, followed by an 8-digit number), whether the probe targets the top or bottom strand (denoted as “T”, or “B”, respectively), whether the probe targets the bisulfite converted strand or complementary strand after amplification (denoted at “C” or “O”, respectively), the Infinium probe design type (Type I probes are denoted as “1”, while Type II probes are denoted as “2”), and the number of times the probe was synthesized for array representation (denoted using a numeric number that is greater than zero).
Examples of locus target identifiers are shown below:
Regarding an example of an IlmnID: cg12345678_TC13.
This probe would be a CG probe with an eight-digit code that relates to the 122mer probe sequence. The eight-digit code and suffixes are independent of genome build revisions. The following “T” indicates that the probe targets the top strand. The adjacent “C” indicates that the probe targets the strand that is initially bisulfite converted. The “1” indicates that the probe has a Type I Infinium design. The “3” indicates that the probe was synthesized three times for representation on the array.
For more information, see the Infinium Mouse Methylation BeadChip Manifest File Release Notes in Product Files.
The product files section of the Infinium Mouse Methylation BeadChip Support Site contains a number of IDAT files that can be downloaded and analyzed using GenomeStudio and other third-party analysis software.
The IDAT files include those for DNA input titrations for B16 and NIH3T3 cell lines, replicates for ApcMin and Mlh1 -/- strain derived tumor DNA, DNA derived from mouse spleen and liver tissue, samples with an increasing amount of DNA methylation, and samples where different proportions of human and mouse DNA have been mixed prior to application on the Mouse Methylation BeadChip.
For more information, see the Infinium Mouse Methylation Demo Dataset in Product Files.