Infinium ffpe qc kit

For all SSAD backgrounds and SSAs, 1.0 μg of extracted DNA was submitted to Macrogen, Inc. (Seoul, South Korea) for methylation microarray. DNA quality control used the Infinium FFPE QC Kit (Illumina, San Diego, USA), DNA restoration used the Infinium HD FFPE DNA Restore Kit (Illumina, San Diego, USA), bisulfite conversion used the EZ-96 DNA Methylation Kit (Zymo Research, Irvine, USA) and methylation microarray used the Infinium MethylationEPIC BeadChip Kit (Illumina, San Diego, USA).

Technical steps were performed on the Integragen platform (Integragen Genomics, Evry, France). The DNA extraction was performed using the QIAamp DNA FFPE Tissue Kit and the Qiacube (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Extracted DNA (250–500 ng) was bisulfite-converted using a Zymo EZ DNA methylation kit and ZR-96 DNA Clean and Concentrator-5 (Zymo Research, Irvine, CA, USA). Bisulfite DNA was processed using the Illumina Infinium HD FFPE DNA Restore kit and Infinium FFPE QC kit (Illumina, San Diego, CA, USA). The standard quality controls confirmed DNA quantity/quality and bisulfite conversion. The DNA was then processed using the Illumina Infinium HumanMethylation EPIC Bead-Chip array (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. The iScan control software was used to generate raw data files from the BeadChip in IDAT format, which were analyzed using GenomeStudio version 2.0 (Illumina, San Diego, CA, USA) and checked for quality measures according to the manufacturer’s instructions.

DNA was isolated from all eight whole blood samples using the BioRobot EZ1 (Qiagen) system according to the manufacturer’s instructions. We provided 1.0 µg of extracted DNA to Macrogen, Inc. (Seoul, South Korea) for methylation microarray analysis. DNA quality control was confirmed using the Infinium FFPE QC Kit (Illumina, San Diego, CA, USA), and DNA restoration was performed using the Infinium HD FFPE DNA Restore Kit (Illumina). Bisulfite conversion was performed using the EZ-96 DNA Methylation Kit (Zymo Research, Irvine, CA, USA), and a methylation microarray was performed using the Infinium MethylationEPIC BeadChip Kit (Illumina, USA). The iScan system (Illumina) was used to read the BeadChips.
Array data were exported, processed, and analyzed using Illumina GenomeStudio version 2011.1 (Methylation Module version 1.9.0) and R version 4.0.3. Each methylation data point was represented by fluorescent signals from methylated (M) and unmethylated (U) alleles. Thereafter, the ratio of fluorescent signals was computed from two alleles as β = (max(M, 0))/(|U| + |M| + 100). Raw β-values were extracted as 865,918 CpGs. Furthermore, background correlations and dye bias equalization were made using the lumi package in R. Beta-mixture quantile normalization was performed to reduce the assay bias using the BMIQ package in R.

The quality of DNA from FFPE blocks was checked using an Infinium FFPE QC Kit (Illumina, San Diego, CA, USA) by triplicate quantitative polymerase chain reaction using 1 ng DNA. The ΔCq was calculated by subtracting the average Cq value of the interrogated sample from the Cq value of the standard sample provided by the manufacturer. All 23 FFPE samples showed ΔCq values of less than 5, which is the recommended threshold for suitability for FFPE restoration.

DNA was isolated using the QIAamp FFPE Tissue kit (Qiagen, Hilden, DE) according to the manufacturer’s instructions. It is known that FFPE samples generally perform poorly on array-based applications due to the highly degenerated DNA. Therefore, the quality of the DNA was verified using the Infinium FFPE QC kit (Illumina Inc., San Diego, CA, USA) according to the manufacturer’s protocol. Only samples with good amplification for all replicates and a maximal ΔCq (difference in quantification cycles compared to the standard) below 5 were selected for use in the bisulfite conversion and restoration step. Bisulfite conversion was performed using the EZ DNA Methylation kit (Zymo Research, Freiburg im Breisgau, DE), according to the manufacturer’s instructions. The array-specific incubation program was used for all samples. After bisulfite conversion, DNA samples were restored using the Infinium HD FFPE Restoration kit (Illumina Inc.).