The Illumina HumanMethylation27 BeadChip array was performed at the Johns Hopkins Stanley Kimmel Comprehensive Cancer Center DNA sequencing core, per the manufacturer's instructions. The arrays were performed in a single batch. We applied empirical Bayes moderated t-statistics implemented in the R/Bioconductor package LIMMA [19 (link)] to compare DNA methylation beta values for probes in CpG Islands between the 7 fusion positive tumors or 7 fusion negative tumors to the set of normal samples. We then identified those genes with FDR adjusted p-values below 0.05 as gene targets and proceeded with further validation.
Humanmethylation27 beadchip array
Manufactured by Illumina
The HumanMethylation27 BeadChip Array is a lab equipment product by Illumina. It is a microarray-based platform designed to analyze DNA methylation patterns across the human genome. The core function of this product is to provide a high-throughput method for quantifying the DNA methylation status of CpG sites within the human genome.
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4 protocols using humanmethylation27 beadchip array
1
Differential DNA Methylation Analysis in Cancer
2
Placental Epigenetic Profiling
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Epigenetic analyses were conducted using placenta from a subset of deliveries (n = 197). Placental samples were obtained within 2 h of delivery, and DNA methylation levels were measured using the Human-Methylation-27 BeadChip array (Illumina Inc., San Diego, CA). Details of these protocols have been previously described [14 (link)].
3
Genome-Wide DNA Methylation Analysis
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To analyze global DNA methylation patterns, 1 µg of genomic DNA was subjected to bisulfite conversion using the EZ DNA Methylation Kit from Zymo Research (Irvine, California). Bisulfite-converted DNA was analyzed for methylation at 27,578 CpG sites using the Illumina (San Diego, California) HumanMethylation27 BeadChip Array according to the manufacturer’s protocol. Signal intensity from methylated and unmethylated probes for all sites was scanned on the Illumina BeadArray Reader, and preprocessed using Illumina GenomeStudio software. The methylation status of individual CpG sites was verified by pyrosequencing. Bisulfite-modified DNA was amplified by PCR using biotin-labeled primers specific for the CDKN2B, MGMT, and CARD10 promoters. The primer sequences for CDKN2B and CARD10 are shown in Table S1 ; primers for MGMT (assays ASY514FS and FS1) were obtained from EpigenDx (Hopkinton, Massachusetts). The biotinylated PCR product was then bound to beads, washed through the Vacuum Prep Tool (Qiagen, Valencia, California), and mixed with sequence-specific primers before analysis on the Pyrosequencer (Qiagen).
4
DNA Methylation Data Integration and Preprocessing
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Data sets were obtained from GEO. All three sets (accession numbers GSE20712, GSE31979, and GSE32393) represent DNA methylation data assayed via the Illumina HumanMethylation27 BeadChip Array platform. Beadchip identifiers were available for data sets GSE20712 and GSE32393, so these were used to preadjust logit-average betas for BeadChip batch effects using the ComBAT algorithm,35 (link) protecting histology, and ER status against over-adjustment. As beta values are required for the method published by Houseman et al.16 (link) adjusted values were subsequently transformed back via inverse-logit to the average beta scale. Only CpGs having fewer than three missing values were used in analysis. We attempted to minimize the size of the models used in this analysis by omitting covariates that displayed only weak overall associations with DNA methylation by preliminary assessment via limma analysis.36 For GSE20712, paired mRNA expression data assayed via Affymetrix Human Genome U133 Plus 2.0 Array exist in GEO via accession number GSE20711; these were used to assign cell-of-origin categories to individual tumors in GSE20712.
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