Genome-wide DNA methylation assays were performed using the HM450K BeadChip (Illumina, Inc., Carlsbad, CA) platform as previously described [48 (link)]. The chips were scanned with Illumina iScan (Illumina, Inc.), and data was extracted using the R package methylumi. The ‘noob’ function in the R package minfi was used to process the data and then the ‘dasen’ function in the R package water melon for normalisation and dye-bias correction. DNA methylation levels were reported as β-value (β = intensity of the Methylated allele/(intensity of the Unmethylated allele + intensity of the Methylated allele)) and calculated using the signal intensity value for each CpG site.
Hm450k beadchip
Manufactured by Illumina
Sourced in United States
The HM450K BeadChip is a laboratory equipment product manufactured by Illumina. It is a microarray-based platform designed for high-throughput analysis of DNA methylation patterns. The core function of the HM450K BeadChip is to provide researchers with a comprehensive tool for interrogating the methylation status of over 450,000 CpG sites across the human genome.
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8 protocols using hm450k beadchip
1
Genome-wide DNA Methylation Profiling
2
Genome-wide DNA Methylation Analysis
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DNA methylation levels of 485 577 CpG (C-phosphate-G) sites were determined using the HM450k BeadChip (Illumina, San Diego, California, USA).19 (link) Signal intensities were parsed into the Minfi R package for quality control (QC) and Subset-quantile Within Array Normalisation.20–22 The post-QC dataset comprised 385 851 CpG sites, 347 patients with SLE and 400 controls for the discovery phase and 201 patients and 188 controls for the replication phase. The aggregate of methylation beta values for all CpG sites followed identical bimodal distributions in both cases and controls (see figure S1 in the online Supplementary file 2 ).
3
DNA Methylation Profiling of Primary Sjögren's Syndrome
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Genomic DNA from whole blood, CD19+ B cells and minor salivary gland biopsies was isolated using standard procedures. DNA methylation levels of 485 577 CpG sites were determined on the HM450K BeadChip (Illumina, San Diego, California, USA). Signal intensities were parsed into the Minfi R package for Subset-quantile Within Array Normalization.20–22 The post-quality control (QC) data set comprised 388 971 CpG sites. To determine differential methylation between patients with pSS and controls a linear regression model containing cell count estimates,23 (link)
24 (link) age and sex as covariates was fitted. DMCs with a Bonferroni-adjusted threshold of p<1.3×10−7 were considered significant.
24 (link) age and sex as covariates was fitted. DMCs with a Bonferroni-adjusted threshold of p<1.3×10−7 were considered significant.
4
Methylation QTLs Near rs2735099 in B Cells
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Previously published data on CpG DNA methylation levels (HM450K BeadChip, Illumina) in B cells from 43 healthy individuals [35 (link)], were analysed for meQTLs within a 1 MB window of rs2735099 using a linear regression model including age and sex as covariates.
5
Genome-wide Methylation Analysis of TCGA Adenocarcinomas
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We analyzed the TCGA dataset generated with the Illumina HM450K Beadchip that interrogated 811 adenocarcinomas and/or squamous cell carcinomas for genome-wide methylation changes. The approach described previously identified 3759 methylated genes with loss of expression ≥ 2-fold for comparison to methylated genes in transformed HBECs [54 (link)].
6
DNA Methylation Analysis of IFN-Regulated Genes
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DNAm was analysed as described previously.15 (link) Briefly, DNAm of 485 577 CpG sites was determined using the HM450K BeadChip (Illumina). Signal intensities were parsed into the Minfi R package16 (link) for quality control and Subset-quantile Within Array Normalisation.17 (link) Methylation beta values were calculated as the fraction of the signal intensity from the methylated CpG sites over the total intensity (range 0–1, corresponding to 0%–100% methylation). DNAm beta values at type I IFN-regulated genes with the strongest correlation with the RNAseq IFN score were identified at cg10549986 (RSAD2), cg05552874 (IFIT1) and cg05696877 (IFI44L) and were used to calculate DNAm IFN scores.
7
DNA Extraction and Illumina Microarray
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DNA was extracted from cells recovering from 2 Gy irradiation at indicated time points using the Qiagen DNeasy kit. Illumina HM450K BeadChips were performed by the Australian Genome Research Facility.
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Illumina DNA Methylation Profiling Workflow
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DNA methylation was evaluated using the Illumina Infinium Human Methylation 450 K BeadChips (HM450K; Illumina)100 (link). Briefly, 1 μg of genomic DNA was bisulfite converted using the EZ DNA Methylation-Direct kit (Zymo Research Irvine, CA). The bisulfite conversion efficacy was evaluated using the MethyLight assay for a panel of defined markers. Samples passing quality control (QC) were whole genome amplified, enzymatically fragmented, hybridized onto HM450K BeadChips, and scanned using the Illumina iScan microarray scanner (Illumina). The raw data (in IDAT file format) was processed with the ‘lumi’ R package using default parameters, and DNA methylation values were calculated as M values for use in subsequent analyses. Annotation of HumanMethylation450 probes was downloaded from the Illumina website (‘https://support.illumina.com/downloads/humanmethylation450_15017482_v1-2_product_files.html ’). Probes associated with individual genes were corrected according to gene location information from GENCODE Human v19. Briefly, probes located within 2000 bp upstream of the gene start site were annotated as ‘TSS2000’ or ‘Promoter’, and probes located between gene start and gene end were annotated as ‘GeneBody’. Existing annotations of 5’UTR or 3’UTR were retained. DNA methylation data were not available for two cell lines in the refined cohort (S2350, S2667).
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