Rna seq alignment workflow

RNA was isolated from formalin-fixed paraffin-embedded (FFPE) tissue sections using RNeasy FFPE Kit of Qiagen (Hilden, Germany) and quantified spectrophotometrically using NanoDrop-1000 (Waltham, United States). Molecular analysis was performed using the TruSight RNA Fusion Panel (Illumina, Inc., San Diego, CA, USA) with 500 ng RNA as input according to the manufacturer’s protocol. Libraries were sequenced on a MiSeq (Illumina, Inc., San Diego, CA, USA) with >3 million reads per case, and sequences were analyzed using the RNA-Seq Alignment workflow, version 2.0.1 (Illumina, Inc., San Diego, CA, USA). The RNA-Seq alignment app (Illumina) was employed to call fusions by using the TopHat-Fusion algorithm, and to generate raw counts for each of the targeted 507 genes. Additionally, the Integrative Genomics Viewer (IGV), version 2.2.13 (Broad Institute, REF) was used for data visualization of fusions. For samples 6, 8, and 11, the fusion calling algorithm revealed a failure of quality control parameters, and no fusion was called. However, visual inspection of the automatically generated bam files revealed an HMGA2-NCOR2 gene fusion in sample 6 at low coverage, but no fusion in samples 8 and 11.

Sequencing was performed using the NextSeq 500/550 High-Output v2 (150 cycle) Kit on the NextSeq 550 platform. The Illumina RNA-Seq Alignment Workflow was used to generate count files (genome assembly was performed (hg19; Illumina)). Gene expression level normalization was performed by DESeq2 (v.1.26.0, Bioconductor v.3.10)^{77 (link)} which was used for downstream analysis. Pathway analysis of gene identifiers extracted from RNA-seq was performed using Ingenuity Pathway Analysis (Ingenuity Systems, www.ingenuity.com, Qiagen). Volcano plots and heat maps to visualize significantly differentially expressed transcripts (adjusted P value < 0.05, log₂(fold change) > 2) were generated using Prism v.9 (GraphPad). RNA-seq data generated this study have been uploaded to ArrayExpress (E-MTAB-101123; details in Source data for Fig. 1).