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 the Infinium FFPE QC Assay and Infinium HD FFPE DNA Restore kits. This bulletin 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.


2. Evaluate sample quality (FFPE only). For human samples, check DNA quality using the Infinium 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 (refer to the QC protocol).


3. Perform bisulfite conversion of all sample types. Recommended DNA input for Zymo Bisulfite Conversion kit (refer to the approved kits in Table 1):
   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.
    
   Follow the Zymo bisulfite conversion protocol for catalog numbers D5004, D5001, or D5002 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. 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.


5. Proceed with the Infinium Methylation Assay Amplify DNA step.
   Fresh/frozen: Use 4 µl bisulfite-converted DNA input for the Infinium HD Methylation 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. 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: Select MSA4 Tasks in Illumina Automation Control (IAC) software and proceed to Hyb Multi-BC2
     • For MethylationEPIC BeadChip, select the eight-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 1. 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 DNA samples.

For additional information, refer to:

• Infinium Methylation EPIC BeadChip Support Resources Page
• Infinium Mouse Methylation BeadChip Support Resources Page
• Methylation Bulletins
  • Which bisulfite conversion kit should I use for the Infinium Methylation arrays?
  • Infinium MethylationEPIC Manifest Column Headings
  • Infinium Mouse Methylation Beadchip manifest column headings
• Methylation Recorded Webinars
  • Infinium Methylation: Introduction to GenomeStudio analysis and BeadArray Controls Reporter