title: Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations.
abstract: RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding RNAs. Standard RNAseq requires 30 - 100 million sequencing reads and can include multiple RNA products such as mRNA and noncoding RNAs. We demonstrate how targeted RNAseq (capture) permits a focused study on selected RNA products using a desktop sequencer. RNAseq capture can characterize unannotated, low, or transiently expressed transcripts that may otherwise be missed using traditional RNAseq methods. Here we describe the extraction of RNA from cell lines, ribosomal RNA depletion, cDNA synthesis, preparation of barcoded libraries, hybridization and capture of targeted transcripts and multiplex sequencing on a desktop sequencer. We also outline the computational analysis pipeline, which includes quality control assessment, alignment, fusion detection, gene expression quantification and identification of single nucleotide variants. This assay allows for targeted transcript sequencing to characterize gene expression, gene fusions, and mutations.
authors: Department of Internal Medicine, Division of Medical Oncology, Comprehensive Cancer Center, The Ohio State University.
authors: Dorrelyn P
authors: DP
authors: Martin
authors: Department of Internal Medicine, Division of Medical Oncology, Comprehensive Cancer Center, The Ohio State University.
authors: Jharna
authors: J
authors: Miya
authors: Department of Internal Medicine, Division of Medical Oncology, Comprehensive Cancer Center, The Ohio State University.
authors: Julie W
authors: JW
authors: Reeser
authors: Department of Internal Medicine, Division of Medical Oncology, Comprehensive Cancer Center, The Ohio State University; Department of Pharmacology, The Ohio State University; sameek.roychowdhury@osumc.edu.
authors: Sameek
authors: S
authors: Roychowdhury
mesh_terms: D001483:Base Sequence; D020869:Gene Expression Profiling / Q000379:methods; D015894:Genome, Human; D023281:Genomics / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D012313:RNA / Q000032:analysis / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D012335:RNA, Ribosomal / Q000032:analysis / Q000235:genetics; D017423:Sequence Analysis, RNA / Q000379:methods*
chemical_list: D012333:RNA, Messenger; D012335:RNA, Ribosomal; D012313:RNA