Infinium hd methylation protocol

Genomic DNA (gDNA) was extracted from 10 pairs of human CRC tissues and adjacent normal tissues using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol. DNA quality assessment and quantification were performed using the NanoDrop 1000 (Thermo Scientific). Bisulphite conversion was performed according to the manufacturer's recommendations for the Illumina Infinium Assay (EZ DNA methylation kit, ZYMO, Orange, CA). The converted and amplified gDNA fragment was used for hybridization with the 15-mers specific capture probe (Table 2) according to the Illumina Infinium HD methylation protocol for genomic facilities (Novogene, Beijing, China). The nucleotide substrates (A/T and C/G) were labeled by different fluorescent dyes and only the probe with complementary binding of gDNA could be single base extended. Finally, iScan software was used to read and output the methylation level results according to the fluorescence.

The Illumina HumanMethylation450 BeadChip and HiScan reader were used to perform the epigenome-wide methylation analysis. The EZ-96 DNA Methylation^™ Kit (Zymo Research, Orange, CA) was used for bisulfate conversation with 1 μg of input DNA (at 45 μl). 4 μl of bisulfite-converted DNA were used for DNA methylation assays, following the Illumina Infinium HD Methylation protocol. This consisted of a whole genome amplification step followed by enzymatic end-point fragmentation, precipitation, and resuspension. The resuspended samples were hybridized on HumanMethylation 450 BeadChips at 48°C for 16 h. The individual samples were assigned to the BeadChips and to chip position using the same sampling scheme as that for the expression BeadChips.

Bisulfite-conversion of 500 ng of DNA was performed according to the manufacturer’s recommendations (EZ DNA Methylation Gold Kit, Zymo Research, Irvine, California) for the Illumina Infinium Assay. The incubation profile was 16 cycles at 95 °C for 30 s, 50 °C for 60 min and a final holding step at 4 °C for 10 min. In total, 10 μl of bisulfite-converted DNA were used for hybridization on Infinium HumanMethylation 450 BeadChip with 485,557 probes, following the Illumina Infinium HD Methylation protocol (Illumina, Inc, San Diego, California). All arrays passed Illumina standard quality control metrics and were included in this study.

Based on the similarities between human and dog and on the concepts of homology and orthology, Infinium HM850 BeadChip DNA methylationEPIC analysis (Illumina, California, USA) was chosen, representing the methylation state of over 850 K CpG sites [41 (link)]. This platform interrogates 853,307 positions of methylation by sample, located in enhancer regions, body gene, and intergenic regions [42 (link)]. DNA aliquots extracted in the HPLC procedure were used. The quality and quantity of DNA were evaluated by the Quant-iT Picogreen ds DNA test and measured on Qubit fluorometer (Life Technologies, NY, USA). 200 ng of DNA was bisulfite converted with the EZ-96 DNA Methylation-Gold Kit, used according to the manufacturer’s protocol (Zymo Research, Orange, CA, USA). Briefly, 3μl of bisulfite converted DNA were denatured, neutralized, and amplified; after precipitation with isopropanol, the DNA was collected by centrifugation and resuspended in buffer for hybridization to HM 850 BeadChip at 48°C for 16 h, followed by single nucleotide extension according to the Illumina Infinium HD Methylation protocol. Next, the incorporated nucleotides were labelled with biotin (ddCTP and ddGTP) and 2,4-dinitrophenol (DNP) (ddATP and ddTTP), and the BeadChip was then scanned using a Illumina HiScan scanner (Illumina, San Diego, CA, USA).

DNA methylation profiles were generated at AbbVie, Inc. from blood samples of ADNI participants using Infinium® MethylationEPIC BeadChip (Illumina, Inc., San Diego, CA, USA). Genomic DNA samples obtained from National Cell Repository for Alzheimer’s Disease were bisulfite-converted using the EZ-DNA Methylation Kits (Zymo Research, Irvine, CA, USA) and subsequently analyzed using the Illumina Infinium® HD methylation protocol on the HiScan™ system (Illumina, Inc). Detailed sample-level quality control was described previously [20 (link)]. A probe-level quality control was performed as well; probes that did not perform well (probes with detection P value ≥ 0.05 in ≥ 1% samples [n = 3047] and probes with bead count < 3 in ≥ 5% of samples [n = 1011]) were filtered out. A total of 863,718 probes were used in the downstream analysis. Methylation profiles from 653 baseline DNA samples were used in this study. Participants with reverse conversion (e.g., from CN to MCI and back to CN) or conversion from CN to AD, those with a diagnosis of AD at baseline, and those without post-baseline measurements were excluded, leaving a total of 519 samples reported in the study.