Infinium 850k methylation array

CpG methylation analysis was performed using Infinium 850k Methylation array (Illumina) according to the manufacturer’s recommendations. Briefly, 200 ng of DNA extracted from each sample was utilized to restore the degraded FFPE DNA to a state that is analyzable by the Infinium HD FFPE whole genome amplification workflow as described in the Infinium FFPE Restoration guide (Illumina). DNA was denatured using 8 µL of NaOH 0.1N for 10 min at room temperature. A 1 h reaction at 37°C was then performed with Primer Pre-Restore (PPR) and Amplification Mix Restore (AMR) reagents supplied by the kit manufacturer in which DNA repair is accomplished. Restored DNA was cleaned using a ZR-96 DNA Clean and Concentrator-5 kit (Zymo Research) following the manufacturer’s protocol. DNA was concentrated between 150 ng/uL of concentration. Between 100 and 200 ng of prepared DNA was used as input for the hybridization on the Illumina 850K Epic BeadChip array and processed according to kit instructions. Data were quality assessed using Illumina Genome Studio software (Illumina), and idat files were uploaded to the DKFZ Heidelberg Classifier v12.5 (www.molecularneuropathology.org).^{16 (link)}

We collected publicly available datasets of multi-omics in lung adenocarcinoma (LUAD) from The Cancer Genome Atlas Project (TCGA) and Clinical Proteomic Tumor Analysis Consortium (CPTAC) (Gillette et al. 2020 ). In TCGA, we included tumor and normal patients with transcriptomics, epigenetics, genomics and clinical data. The RNA-seq data (Illumina TruSeq) were transformed by log2 (FPKM + 1) and methylation were Illumina Infinium 450 K methylation array, average promoter methylation was defined as − 1,500 bp to + 500 bp windows relative to the transcription start site. In CPTAC, we included tumor patients with transcriptomics, epigenetics and clinical data. The RNA-seq data (Illumina TruSeq) were transformed by log2(RPKM) and methylation were Illumina Infinium 850 K methylation array. Gene expression was illustrated by plotting the median expression value when the genes were with one more probe. Genes with more than 50% equal to 0 value were deleted, and KNN imputation was imputed the remaining missing values.