Infinium humanmethylation450 beadchip array hm450k

The TCGA database of head and neck cancers was accessed for publically available methylation data assessed by the Infinium HumanMethylation450 beadchip array (HM450K, Illumina inc., San Diego, US).
Methylation data from Level 1 raw IDAT files were obtained from the heterogeneous TCGA HNSCC samples (http://cancergenome.nih.gov/; HM450K download date 24 June 2013; clinical data download date 22 July 2013). Utilizing R software (version 3.0.1; http://cran.r-project.org/), background correction and probe specific correction with SWAN normalization was performed utilizing Methylumi and Minfi packages found in Bioconductor [112 (link)–114 (link)]. Probes mapping to common variant SNPs or non-unique mappings were removed from the analysis, leaving 330,557 probes for the downstream analysis [115 (link)]. Our MS-HRM amplicons interrogating the APC, BRCA1, CDH1, CDH13 and MLH1 genes, overlapped with CpG probes assessed by the HM450K. For the seven genes (ABO, ATM, DAPK1, RASSF1A, MGMT, ERCC1 and RUNX3) that did not have overlapping CpGs on the HM450K, the two probes immediately flanking upstream and downstream of our MS-HRM amplicon were assessed. A β-value greater than 0.2 was used to define the presence of significant methylation [116 (link)].

Melbourne cohort: Genome‐wide DNA methylation was measured on bisulfite‐converted genomic DNA using the Illumina Infinium HumanMethylation450 BeadChip Array (HM450K) at ServiceXS (the Netherlands). Cambridge/Heidelberg cohort: Genome‐wide DNA methylation was measured on bisulfite‐converted genomic DNA using the HM450K array at the German Cancer Research Centre Genomic and Proteomics Core Facility. Raw methylation data from both cohorts were combined for analysis. Data were processed using the lumi and minfi packages for R and normalised using SWAN (Maksimovic et al., 2012). Probes on the X and Y chromosomes, associated with single nucleotide polymorphisms (minor allele frequency > 1%), or which failed in one or more samples were removed, leaving data for 422 877 probes common to all samples for subsequent analysis. Probable BRAF fusion status was predicted by manual inspection of copy number profiles generated from HM450K data by the conumee package for R. Control brain: raw HM450K array IDAT files for 14 snap‐frozen control adult brain tissues were obtained from D.T.W.J. Tissues were from the cerebellum, hypothalamus and cerebral hemispheres. Data were processed as above, which yielded data for 453 805 probes.

We downloaded the level 3 DNA methylation data generated by the Illumina Infinium HumanMethylation450 BeadChip Array (HM450K) for BC patients from The Cancer Genome Atlas (TCGA GDC) database [29 (link)], and calculated Transcripts Per Kilobase Million values (TPM) for the mRNA expression profiles from the UCSC Xena database. The PAM50 subtype information and clinical information was downloaded from cBioPortal database [30 (link)]. We obtained 349/150 female BC luminal A/B samples, 343/542 non-luminal A/B samples based on the PAM50 subtype information and 96 normal samples for subsequent analysis. The non-luminal A/B are other BC subtypes with luminal A/B removed. Survival data used the standardized data set named TCGA Pan-Cancer Clinical Data Resource (TCGA-CDR) [31 (link)] recommended clinical outcome endpoint data for BC. In addition, GSE72308 [32 (link)] was used as an external validation dataset and GSE180286 single-cell sequencing data from The Gene Expression Omnibus (GEO) database. The 1793 immune genes were downloaded from the ImmPort database (https://www.immport.org/shared/home (accesed on 27 February 2018)).