Chapter 24 - RNA-seq: the premier transcriptomics tool

Abstract

RNA-seq is an amalgam of standard molecular biology techniques and computational bioinformatics methods that are designed to identify and quantify transcriptional activity in the cell. The nomenclature, RNA-seq, comes from “RNA sequence analysis” and the technology is a widely regarded asset within the scientific community. The utility and popularity of this transcriptomics tool are based on the high-throughput massively parallel character of NGS and the unique characteristics of third-generation sequencing. RNA-seq has revolutionized and invigorated the mining of the transcriptome for its secrets by providing much greater latitude in experimental design and providing an understanding of the transcriptome at heretofore unknown levels of detail. RNA-seq produces a quantitative and a qualitative profile of each sample. The quantitative profile reflects the expression level of each gene and permits the discernment of abundance relationships among RNAs in the sample across a wide dynamic range. The qualitative data that are generated are essential for observing structural alterations in transcripts as a consequence of transcriptional or posttranscriptional events. While earlier molecular biology hybridization-, PCR-, or microarray-based methods were limited to the study of only one or a few transcripts in a single experiment, RNA-seq will assay whatever transcripts are present in the starting material. Further, no a priori knowledge or designation of target sequences is needed. RNA-seq consists of multiple well-defined steps. The amount of data generated at the end of the sequencing run is enormous and relies on computational bioinformatics software to sort, manage, and mine the data. Much like other standard molecular biology methods, there are multiple variants of PCR that focus on different RNA subpopulations or on RNA molecules which exhibit specific characteristics.