Introduction

The Illumina Infinium EX Methylation Assay transforms DNA Methylation analysis by streamlining sample preparation and enabling high-volume sample processing. The assay scales methylation profiling to hundreds of thousands of CpG loci per sample and supports the processing of large sample numbers with automation.

The Infinium EX 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, 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 EX Methylation, DNA is treated with sodium bisulfite using an Illumina-recommended bisulfite conversion kit. The treatment converts unmethylated cytosines to uracils, while methylated cytosines are protected from conversion by the methyl group and remain as cytosines.

The sample DNA is then entered into the Infinium EX Methylation. A WGA reaction converts the uracils generated during bisulfite conversion to thymines while amplifying the samples several hundred-fold. The amplified samples are fragmented through an endpoint enzymatic reaction, and the fragmented DNA is hybridized to the probes on the Infinium 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 EX Methylations 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 which probe (methylated or unmethylated) has been extended by the labeled base to discern which allele is present.

This resource provides instructions for performing the semi-automated assay workflow using the following systems:

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Infinium Automated Pipetting System (IAPS) with Illumina Lab Automation Software Solution (ILASS)

The Infinium EX Methylation Assay for Methylation offers the following features:

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PCR-free amplification.

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Minimal risk of carryover contamination.

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Multiple-sample BeadChip format.

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Improved reagent chemistry.

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Low DNA input into bisulfite conversion.

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50 ng to 1 µg input for the manual process on fresh DNA (higher input results in higher reproducibility between samples)

Best Practices

All Infinium EX Methylation Assay documentation requires that you have set up the laboratory space, are familiar with the standard procedures and safety precautions, and understand how to use the IAS. For more information, refer to the following resources:

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Infinium Lab Setup and Best Practices

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Infinium Assay Consumables and Equipment List

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Infinium RoMa (IAPS with ILASS) Product Documentation

Before hybridization, download the decode map (DMAP) files for the arrays. Refer to the Illumina Support Site to download the DMAP files from the Decode File Client.

System Maintenance and Usage

IAPS with ILASS

Before beginning an automated run, complete daily maintenance tasks and perform a system flush. Repeat the system flush if the instrument is idle for two hours or longer (on the same day). Perform a bleach wash at the beginning or the end of each day. For more information, refer to the Infinium RoMa (IAPS with ILASS) Product Documentation.