DNA Input Guidelines for Infinium MethylationEPIC Arrays

General Infinium assay sample input recommendations established for genotyping arrays are broadly applicable to methylation arrays. These include guidance on DNA quality, integrity, and preparation. However, the required DNA input amount for methylation arrays differs and must be considered separately.

For comprehensive guidance on sample preparation requirements, refer to Guidelines for Sample Preparation for Infinium Genotyping Assay DNA input .

For MethylationEPIC Arrays, the minimum DNA input for the bisulfite conversion reaction is 250 ng. For optimal results and higher reproducibility, Illumina recommends at least 500 ng and up to 1000 ng.

Table 1. Minimum DNA Input Recommended by Assay and Sample Type

Workflow	Minimum DNA input
Manual	250 ng to 1000 ng
Automated	1000 ng
FFPE	250 ng

For additional details on the Infinium Methylation workflow refer to the following resources:

• Infinium HD Methylation assay workflow for fresh/frozen versus FFPE samples

• Infinium HD Methylation Assay Reference Guide

These resources provide comprehensive guidance on workflow steps, sample type considerations, and assay-specific requirements.

Recommended DNA Sample Volumes for Zymo Bisulfite Conversion

Illumina has validated and recommends the use of specific Zymo Bisulfite Conversion kits for Infinium Methylation Assays.

For further information on selecting an appropriate kit, refer to the Illumina Knowledge on Which bisulfite conversion kit should be used for the Infinium Methylation arrays?

For Zymo kits D5001, D5002, and D5004, perform DNA preparation according to the manufacturer’s protocol as follows:

1. Add 5 µl of M-Dilution Buffer to the DNA sample.

2. Adjust the total reaction volume to 50 µl with nuclease-free water.

3. Mix the sample thoroughly by gentle flicking or by pipetting up and down.

Example: For a DNA sample volume of 14 µl, add 5 µl of M-Dilution Buffer and 31 µl of water to achieve a total volume of 50 µl.

This table outlines the component volumes required for preparation of the bisulfite conversion reaction. A fixed volume of 5 µL M-Dilution Buffer is used, and the final reaction volume must be adjusted to 50 µL. Accordingly, up to 45 µL of DNA (corresponding to an input range of 250-1000 ng) may be used, with nuclease-free water

Table 2. Reaction setup for Zymo bisulfite conversion using M-Dilution Buffer

Component	Volume
M-Dilution buffer	5 μl
DNA	variable
Water	variable
Total	50 μl

For Zymo kits D5046 and D5047, perform DNA preparation according to the manufacturer’s protocol as follows:

1. Add 130 µl of Lightning Conversion Reagent to 20 µl of DNA sample in a Conversion Plate.

2. Mix the samples thoroughly by pipetting up and down.

Note: If the DNA sample volume is less than 20 µl, adjust to the required volume with nuclease-free water before adding the Lightning Conversion Reagent.

For Zymo Kit D5049, perform DNA preparation according to the manufacturer’s protocol as follows:

1. Add 130 µl of Lightning Conversion Reagent to20 µl of purified DNA sample in a non-skirted 96-well microplate (user supplied).

2. Mix the sample thoroughly by pipetting up and down.

Note:

• DNA quantification must be performed prior to bisulfite conversion. Following bisulfite conversion, DNA quantification is not required, and the user may proceed directly to the Infinium Methylation Assay.

For any feedback or questions regarding this article (Illumina Knowledge Article #2679), contact Illumina Technical Support [e-mail].