Finally, RNA-seq can be coupled with different types of biochemical assay to analyze many other aspects of RNA biology, such as RNA–protein binding, RNA structure, or RNA–RNA interactions. RNA-seq can be used solo for transcriptome profiling or in combination with other functional genomics methods to enhance the analysis of gene expression. Both the experimental design and the analysis procedures will vary greatly in each of these cases. Furthermore, investigators might be interested only in messenger RNA isoform expression or microRNA (miRNA) levels or allele variant identification. For well-annotated genomes such as the human genome, researchers may choose to base their RNA-seq analysis on the existing annotated reference transcriptome alone, or might try to identify new transcripts and their differential regulation. By contrast, for organisms without sequenced genomes, quantification would be achieved by first assembling reads de novo into contigs and then mapping these contigs onto the transcriptome. For example, if a genome sequence is available for the studied organism, it should be possible to identify transcripts by mapping RNA-seq reads onto the genome. Scientists plan experiments and adopt different analysis strategies depending on the organism being studied and their research goals. There is no optimal pipeline for the variety of different applications and analysis scenarios in which RNA-seq can be used. Many variations of RNA-seq protocols and analyses have been published, making it challenging for new users to appreciate all of the steps necessary to conduct an RNA-seq study properly.

The pervasive adoption of RNA-seq has spread well beyond the genomics community and has become a standard part of the toolkit used by the life sciences research community. The power of sequencing RNA lies in the fact that the twin aspects of discovery and quantification can be combined in a single high-throughput sequencing assay called RNA-sequencing (RNA-seq). Transcript identification and the quantification of gene expression have been distinct core activities in molecular biology ever since the discovery of RNA’s role as the key intermediate between the genome and the proteome.

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