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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