Illumina whole genome transcriptomic RNA sequencing of sample 1703
Following RNA extraction (RNeasy, Qiagen), library construction was performed using a non-strand specific Illumunia TruSeq protocol. Flowcell cluster amplification and sequencing were performed according to the manufacturer’s protocols using HiSeq 2000/2500, with a 76 bp paired-end run including an eight-base index barcode read. RNA-sequencing files were downloaded from published datasets [Zhang J et al.,Nat Gen,2013; Jones DTW et al.,Nat Gen,2013]. RNA-seq bam files were transformed to fastq files using the Picard SamToFastq algorithm. Raw paired-end reads were aligned to the reference genome hg19 and preprocessed using PRADA (Pipeline for RNA-sequencing Data Analysis) [Torres-Garcia et al.,Bioinformatics,2014]. We used PRADA within Firehose to determine gene-expression levels, exon expression levels, quality metrics, and for detection of fusion transcripts. BAM files were also assessed by visual inspection with IGV [Robinson JT et al.,Nat Biotech,2011].