Whole transcriptome sequencing data processing
hybridization/capture, tumor
TUMOR
Whole transcriptome sequencing (RNA-seq) was performed on bulk RNA extracted from 75 baseline tumor tissue samples at Q Squared Solutions Expression Analysis. Sequencing libraries were generated using the Illumina TruSeq RNA Access method, a hybridization-based protocol to enrich for coding RNAs from total RNA-seq libraries. The method consists of two major steps: total RNA library preparation and coding RNA library enrichment. First-strand complementary DNA synthesis was primed from total RNA using random primers, followed by the generation of second-strand cDNA with dUTP (in place of dTTP) in a master mix. This facilitates the preservation of strand information, as amplification in subsequent steps will stall when it encounters uracil in the nucleotide strand. Double-stranded cDNA then underwent end repair, A-tailing and ligation of adaptors that include index sequences. One sample failed library preparation and was removed from the dataset. The resulting molecules were amplified via PCR, their yield and size distribution were determined using a BioAnalyzer and their concentrations were normalized in preparation for the enrichment step. Libraries were enriched for the messenger RNA fraction by positive selection using a cocktail of biotinylated oligonucleotides corresponding to coding regions of the genome. Targeted library molecules were then captured through the hybridized biotinylated oligonucleotide probe using streptavidin-conjugated beads. After two rounds of hybridization/capture reactions, the enriched library molecules were PCR amplified, quantified, then normalized and pooled in preparation for sequencing on an Illumina HiSeq 2500. Libraries were sequenced using the Illumina sequencing-by-synthesis platform on an Illumina HiSeq 2500 at a paired-end read length of 50 bp and a total read depth of 50 million reads per sample. Seven samples failed sequencing quality control and were removed from further analysis.RNA-seq raw reads were aligned to the human genome reference (hg38) using STAR aligner v.2.4.2a with default settings