LIMS support is available for the Infinium HumanMethyation450 BeadChip and Infinium MethylationEPIC BeadChip.
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.
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.
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.
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.
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.
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.
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.
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.
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:
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.
Yes. Illumina recommends the following of-the-shelf bisulfite conversion kits from Zymo Research.
D5001 50 reactions (single-column format)
D5002 200 reactions (single-column format)
D5004 2×96 reactions EZ-96 DNA methylation kit (deep-well format)
Yes, the same controls types are present.
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:
Samples from the same individual:
Samples from different persons:
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.
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.
Similar to the Infinium genotyping assay, we recommend DNA that is sized ≥ 2kb, which can be assessed on an agarose gel.
A beta value (β) is calculated for each CpG locus.
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.
The manifest for the array can be found in the Downloads section of the BeadChip support page.
For more 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.
The beta value (β) uses the ratio of intensities between methylated and unmethylated alleles to estimate the methylation level of the CpG locus.
For Infinium arrays, Illumina recommends use of PicoGreen reagent for DNA quantification. UV-based methods may can overestimate DNA concentration by 2–10 fold.
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.