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


All DNA samples used with the Infinium HD Methylation assay, whether fresh/frozen or formalin-fixed, paraffin-embedded (FFPE), require a Zymo bisulfite conversion kit (with modified protocol). FFPE samples also require FFPE QC Assay and Infinium HD FFPE DNA Restore kits. This bulletin describes the differences in workflow for these sample types.


  1. Isolate DNA using a kit of your choice, then quantify DNA using a fluorometric method (eg, PicoGreen) to ensure accurate measurement of double-stranded DNA.

    Note: Accurate DNA quantification at this step is critical. Following bisulfite conversion, all subsequent steps deal in volumes only and rely on the assumption that DNA input was accurately measured at this point.
  2. FFPE only:Check DNA quality using the Illumina FFPE QC kit and a qPCR kit with green fluorescent dye of your choice. Illumina recommends proceeding only with samples with delta Ct (or delta Cq) < 5 (see QC protocol).
  3. Zymo Bisulfite Conversion kit (see approved kits below) DNA input: Fresh/frozen: > 250 ng (manual) or 1000 ng (automated)
    FFPE:≥ 250 ng (up to 1000 ng)
    *Using > 250 ng of DNA, such as 500 ng, is recommended for the best results and reproducibility, especially for FFPE samples.

    Perform bisulfite conversion of all sample types according to the Zymo bisulfite conversion protocol with the following critical adjustment to the thermal cycler program (end of Day 1), as stated in the Zymo protocol appendix:
    Incubate samples as follows for 16 cycles:
    • 95°C for 30 seconds
    • 50°C for 1 hour
    Then, hold at 4°C until ready to proceed with the Zymo bisulfite conversion protocol for Day 2.

    Elute bisulfite-converted samples (Fresh/frozen and FFPE) in 12 µl (manual) or 22 µl (automated) buffer provided by Zymo.
  4. Fresh/frozen:Use 4 µl bisulfite-converted DNA input for the Infinium Methylation HD protocol, beginning with the steps to make the MSA4 plate.

    FFPE:Use the entire bisulfite-converted eluate (~8 µl) as input for the Infinium HD FFPE DNA Restore kit, and follow the restore protocol. Then use 8 µl restore kit eluate as input in the Infinium HD FFPE Methylation450K protocol (automatedor manual) beginning with the steps to make the MSA5 plate. For MethylationEPIC arrays make sure to Hyb 26 µl, rather than 15 µl,and use guide-E if using automation (see Infinium HD Methylation Assay Protocol Guide).

Additional Kits Required for Infinium HD Methylation:

Product Name Size Catalog
Required for
Required for
Zymo Bisulfite Conversion
Kits approved by Illumina
Methylation™ Kit
(Spin Column Format)*
50 rxn D5001 Yes Yes
Methylation™ Kit
(Spin Column Format)*
200 rxn D5002 Yes Yes
Zymo EZ-96 DNA
Methylation™ Kit
(Deep Well Format)*
2 x 96 rxn D5004 Yes Yes
FFPE Sample QC Illumina FFPE
QC Kit
384 rxn WG-321-1001 No Yes
FFPE Sample Restoration Illumina Infinium HD
FFPE DNA Restore Kit
24 rxn WG-321-1002 No Yes

*Illumina recommends only these Zymo Research Bisulfite Conversion kits
For additional information, see: