Kit Overview
The Illumina Infinium HD Methylation Assay combines bisulfite conversion of genomic DNA and whole-genome amplification (WGA) with array-based capture and scoring of the CpG loci. Signal intensity is measured with the Illumina iScan, NextSeq 550, or NextSeq 550Dx systems to generate beta values, a measure of the relative degree of methylation at a locus. Beta values can be interrogated and compared across samples for powerful, large-scale studies.
Before samples are run through the Infinium Assay, DNA is first treated with sodium bisulfite using an Illumina recommended bisulfite conversion kit. The treatment converts unmethylated cytosines to uracil, while methylated cytosines are protected from conversion by the methyl group and remain as cytosines.
The sample DNA is then entered into the Infinium Assay assay. A WGA reaction converts the uracils generated during bisulfite conversion to thymines while amplifying the samples several hundredfold. The amplified samples are fragmented through an endpoint enzymatic reaction, and the fragmented DNA is hybridized to the probes on the Infinium Assay microarray. Individual CpG loci are interrogated to determine whether the cytosines of the CpG locus were converted to uracil or not by the bisulfite conversion reaction. Detected cytosines indicate that the interrogated site was methylated, while detected thymines indicate that the interrogated site was not methylated.
Individual Infinium Assay assays interrogate their targets with either an Infinium I (two-probe) or Infinium II (single probe) design. The Infinium I design deploys two probes per CpG locus, one unmethylated and one methylated. The 3’ terminus of each probe is complementary to either the cytosine (methylated) or thymine (unmethylated) base at the CpG locus being interrogated. After hybridizing the fragmented sample DNA to the assay probes, the probes extend by a single labeled base. Because the extension reaction requires the 3’ terminal base to be complementary to the hybridized target to take place, one need only track the probe (methylated or unmethylated) extended by the labeled base to discern which allele is present.
Infinium II design deploys a single probe whose 3’ terminus stops one base short of the cytosine site being interrogated. After hybridization, the single base extension reaction adds the base, which is complementary to the existing base at the interrogation site. Because the extension bases are labeled differentially, with the adenine extension base labeled to yield a red signal during scanning, and the guanine base yielding a green signal, one only needs to determine which color, red or green, is detected during scanning to discern which allele (thymine or cytosine) is present, and ultimately, whether the site was methylated.
The Infinium HD Methylation Assay offers the following features:
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Minimal risk of carryover contamination |
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Low DNA input into bisulfite conversion |
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250 ng to 1 µg input for the manual process on fresh DNA (higher input results in higher reproducibility between samples) |
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1000 ng for the automated process |
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For FFPE samples, refer to the Infinium HD FFPE Assay Reference Guide (Document # 15021525) |
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Automation using the Infinium Automated Pipetting System (IAPS) |
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Compatible with Illumina iScan, NextSeq 550, and NextSeq 550Dx Systems |
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Multiple sample BeadChip format |
