SUPPLEMENT: METHODS:
The pathology was reported at Tata Medical Center, Kolkata (LZ and PR). Review and confirmation of the pathology (CH), and further molecular testing were performed at the Hospital for Sick Children (SickKids), Toronto. DNA methylation profiling was done using the Illumina Human Methylation 850 (850k) array. The idat files were uploaded on the molecular neuropathology 2.0 website (https://www.molecularneuropathology.org/mnp) and were analysed on the brain tumor classifier versions 11b4v.3 and 12.3v.1. Further analysis, including the unsupervised clustering, was performed in R v.4.0.3. Unprocessed .idat files for control methylation tumor classes were downloaded from the NCBI Gene Expression Omnibus (GEO), under accession number GSE73801, as published before1. The ChAMP, minfi, limma and Rtsne packages were utilized for downstream analyses. Raw data were obtained from idat files and normalized using the single sample normal-exponential out-of-band method (ssNoob). Probes on the X and Y chromosomes were removed, along with probes with a bead count of less than 3, those with a detection p value above 0.01, and probes at known SNP locations or with multiple binding locations as previously reported by our group.11 T-distributed stochastic neighbor embedding (t-SNE) analysis using the Rtsne package applied the following nondefault parameters: theta = 0, pca = F, max_iter = 7500, and perplexity = 15. Copy number alterations of genomic segments were inferred from the methylation array data based on the R-package conumee after additional baseline correction (https://github.com/dstichel/conumee). Visualization was performed using the ggplot2 and the plots were edited for aesthetics using Adobe Illustrator v.25.4.1.