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 (certain Zymo kits have modified Illumina protocol).

FFPE samples also require the Infinium FFPE QC Assay and Infinium HD FFPE DNA Restore kits. This article describes the differences in workflow for fresh/frozen versus FFPE DNA samples.

1. Isolate DNA using a kit of your choice, then quantify DNA using a fluorometric method (eg, PicoGreen, Qubit) 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.

1. Evaluate sample quality (FFPE only).For human samples, check DNA quality using the Infinium FFPE QC kit (see Support page) and a qPCR kit with green fluorescent dye of your choice. Illumina recommends proceeding only with samples with delta Ct (or delta Cq) < 5 (refer to the QC protocol).

2. Perform bisulfite conversion of all sample types.

Recommended DNA input for Zymo Bisulfite Conversion kit (refer to the approved kits in Table 2):

Fresh/frozen: > 250 ng (manual) or 1000 ng (automated). FFPE: ≥ 250 ng (up to 1000 ng).

Note: Using > 250 ng of DNA, such as 500 ng, is recommended for the best results and reproducibility, especially for FFPE samples. When using Lightning MagPrep kits, the recommended FFPE DNA input is 1000 ng.

Table 1. Comparison of Zymo classic vs Lightning MagPrep bisulfite conversion kits. The classic kits follow a modified protocol.

	Zymo D5004, D5001, or D5002 (classic kits)	Zymo D5046, D5047 or D5049 (Lightning MagPrep kits)
Thermal cycler protocol	Follow 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.	Follow the Zymo bisulfite conversion protocol with no modifications to the thermal cycler program.
Elution	Elute bisulfite-converted samples (Fresh/frozen and FFPE) in 12 µl (manual) or 22 µl (automated) buffer provided by Zymo.	Elute bisulfite-converted samples (Fresh/frozen and FFPE) in 25 µl buffer provided by Zymo.

1. Restore DNA samples (FFPE only). Use the entire bisulfite-converted eluate (~8 µl) as input for the Infinium HD FFPE DNA Restore kit, and follow the restore protocol.

2. Proceed with the Infinium Methylation Assay Amplify DNA step.

Fresh/frozen: Use 4 µl bisulfite-converted DNA input for the Infinium HD Methylation Assay protocol, beginning with the steps to make the MSA4 plate.

FFPE: Use 8 µl restore kit eluate as input in the Infinium HD FFPE Assay with the steps to make the MSA5 plate.

1. After completing the Resuspend DNA step, proceed to Hybridize DNA to the BeadChip.

• Manual protocol.

  • For MethylationEPIC (8x1 BeadChips), make sure to Hyb 26 µl, rather than 15 µl.

  • For Mouse Methylation (12x1 BeadChips), Hyb 15 µl.

• Automated protocol using Tecan/IAPS with IAC: Select MSA4 Tasks in Illumina Automation Control (IAC) software and proceed to Hyb Multi-BC2.

  • For MethylationEPIC BeadChip, select the 8-sample BeadChip picture and use Tip Guide-E for Hybridization.

  • For Mouse Methylation arrays, select the leftmost 12-sample BeadChip picture and use Tip Guide-B for Hybridization.

  • Refer to the Infinium HD Methylation Assay Reference Guide for more information.

Table 2. Additional Kits Required for Infinium HD Methylation: Illumina has validated and recommends only the Zymo Research Bisulfite Conversion kits listed in this table. All kits listed are compatible with fresh/frozen and FFPE DNA samples. *If using the Lightning MagPrep kits, the recommended FFPE DNA input is 1000 ng. For additional information, refer to:* Infinium MethylationEPIC BeadChip Support Resources Page

• Infinium Mouse Methylation BeadChip Support Resources Page

• Illumina Knowledge Articles:

  • Which bisulfite conversion kit should I use for the Infinium Methylation arrays?

  • DNA Input Guidelines for Infinium MethylationEPIC Arrays

• Methylation Recorded Webinars

  • Infinium Methylation: Introduction to GenomeStudio analysis and BeadArray Controls Reporter

For any feedback or questions regarding this article (Illumina Knowledge Article #1566), contact Illumina Technical Support [e-mail].