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[…] Exome capture reads were aligned to the hg19 build of the human genome using bwa v0.7.5a (bio-bwa.sourceforge.net), and PCR duplicates removed with PicardTools 1.5 (pcard.sourceforge.net). Somatic single nucleotide variants were called using the Genome Analysis Tool Kit v3.3-0 based upon current Best Practices using local re-alignment around InDels, downsampling and base recalibration with variants called by the Unified Genotyper (broadinstitute.org/gatk/). Somatic variants were covered by at least 20 reads in both tumor and normal sequences and carried at least 5 ALT reads in the tumor sequence; unmatched exomes (n=3) were annotated by ExAc and ANNOVAR. Variants were annotated using the Ensembl Variant Effect Predictor v74 (ensembl.org/info/docs/variation/vep) incorporating SIFT (sift.jcvi.org) and PolyPhen (genetics.bwh.harvard.edu/pph2) predictions, COSMIC v64 (sanger.ac.uk/ genetics/CGP/cosmic/) and dbSNP build 137 (ncbi.nlm.nih.gov/sites/SNP) annotations. Copy number was obtained by calculating log2 ratios of tumor/normal coverage binned into exons of known Ensembl genes, smoothed using circular binary segmentation (DNAcopy, www.bioconductor.org) and processed using in-house scripts. Mutation signatures were ascertained by grouping somatic substitutions on the basis of their 3′ and 5′ bases into 96 possible trinucleotide categories (). NGS fusion panel alignment was performed against the human reference sequence GRCh37/Hg19. Quality control (QC), variant annotation, deduplication and metrics were generated for each sample. Manta (https://github.com/Illumina/manta) and Breakdancer (breakdancer.sourceforge.net) were used for the detection of structural variants. [...] Methylation data from the Illumina Infinium HumanMethylation450 BeadChip was preprocessed using the minfi package in R. DNA copy number was recovered from combined intensities using the conumee package with reference to methylation profiles from normal individuals provided in the CopyNumber450kData package. We have used the Heidelberg brain tumor classifier () (molecularneuropathology.org) to assign subgroup scores for each tumor compared to 91 different brain tumor entities using a training set built from 2,801 tumors implemented in the MNP R package (v11b2). Simplified methylation subgroup assignments were then made to incorporate cases carrying G34R/V or K27M mutations in H3 histones, IDH1 mutation at R132, low grade glioma-like profiles (predominantly diffuse infantile ganglioglioma and pilocytic astrocytoma) and those similar to pleomorphic xanthoastrocytoma (PXA). Low-scoring cases, or those with a high normal cell contamination were assigned to G34, K27 or IDH1 groups if the respective mutation was identified. Wild-type HGG encompassed many other methylation subgroups and were simply assigned by exclusion with the groups above. Clustering of beta values from methylation arrays was performed using the 10K probeset from the Heidelberg classifier based upon Euclidean distance with a ward algorithm. Methylation heatmaps show only the most variable probes of the classifier between simplified methylation subgroups. [...] RNA sequences were aligned to hg19 and organized into de-novo spliced alignments using bowtie2 and TopHat version 2.1.0 (ccb.jhu.edu/software/tophat). Fusion transcripts were detected using chimerascan version 0.4.5a filtered to remove common false positives. RNASeq raw count files were used to construct an expression matrix using Roche's internal pipeline. The expression matrix was normalized using edgeR and Voom in R (cran.rproject.org/), and a heatmap was created from the absolute gene expression data using Tibco Spotfire, as previously described (). The mean expression of the signature genes was used to compare MAPK-altered to non-altered samples and statistical significance calculated using a two-tailed unpaired t-test. The mean expression was also used to correlate with CD8 positivity by IHC. Gene Set enrichment analysis was performed using the GSEA java application based upon pairwise comparisons of MAPK altered versus wild-type for curated canonical gene sets. All data are deposited in the European Genome-phenome Archive (ebi.ac.uk/ega/home) under accession number EGAS00001002328. […]

Pipeline specifications

Software tools Bowtie2, TopHat, TopHat-Fusion, chimerascan, edgeR, GSEA
Application RNA-seq analysis
Organisms Enterovirus E, Homo sapiens
Diseases Glioma, Lymphoma, Non-Hodgkin, Neoplasms