DNA Input Guidelines for Infinium MethylationEPIC Arrays

General Infinium Assay sample input recommendations for genotyping arrays are broadly applicable to methylation arrays, with the exception of DNA input amount. For further information, see article 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.

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

For additional details on the Infinium Methylation workflow:

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

• Infinium HD Methylation Assay Reference Guide

Recommended DNA Sample Volumes for Zymo Bisulfite Conversion

Illumina has validated and recommends specific Zymo bisulfite conversion kits. For further information, see article Which bisulfite conversion kit should be used for the Infinium Methylation arrays?

For Zymo kits D5001, D5002, and D5004, the Zymo protocols state:

1. Add 5 µl of M-Dilution Buffer to the DNA sample and adjust the total volume to 50 µl with water. Mix the sample by flicking or pipetting up and down. Example: For 14 µl of a DNA sample add 5 µl M-Dilution Buffer and 31 µl water.

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

5 µl of M-Dilution Buffer is required for the reaction and the final volume must be 50 µl, thus up to 45 µl of DNA (250 ng - 1000 ng) can be used as input for the bisulfite conversion.

For Zymo kits D5046 and D5047, the Zymo protocol states:

1. Add 130 µl of Lightning Conversion Reagent to 20 µl of a DNA sample in a Conversion Plate. Mix the samples by pipetting up and down. Note: If the volume of DNA is less than 20 µl, compensate with water.

For Zymo Kit D5049, the Zymo protocol states:

1. Add 130 µl of Lightning Conversion Reagent to 20 µl of a purified DNA sample in a non-skirted 96-well microplate (user provided). Mix thoroughly by pipette mixing.

Note: DNA quantification must be performed before bisulfite conversion. After bisulfite conversion, the DNA does not need to be quantified and the user can 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].