Figure 1. RNA-seq decision tool. Available here.

Helping to clarify RNA-Seq

RNA-Seq is a powerful technique that uses next-generation sequencing (NGS) to obtain sequence reads from millions of individual RNAs. This technique can be used to:

  • Measure gene expression.
  • Characterize alternative splicing, alternative polyadenylation, and alternative transcription.
  • Discover and annotate transcripts including identify their 5’ and 3’ ends and splice-junctions.

RNA-Seq has been widely adopted. However, it is important to realize that the technique is still being actively developed. Currently, numerous variations and adaptations are available. Before undertaking an RNA-seq experiment researchers must choose from the many options that are available. Some of these choices will have little impact on the final results; however, other choices can greatly impact the quality and usefulness of the final data.

Here we attempt to provide an overview of RNA-seq and of the choices that one must make to carry out a successful RNA-seq experiment.

Site overview

The purpose of this site is to provide a comprehensive discussion of each of the steps that are involved in performing RNA-seq, and to highlight the primary options that are available along with some guidance for choosing between various options. The primary content is topically organized according to the work-flow of a typical RNA-seq experiment. Links to these sections can be found under "Experimental Details" in the top menu.

We have also developed a simple interactive decision tool (see figure 1) that highlights recommended options based upon user defined experimental parameters.

RNA-Seq workflow:

There is no one-way to perform an RNA-seq experiment. A survey of literature will reveal many variations in experimental design, sample preparation, and analysis. Why are there so many variations? Some variations are related to the goal or objective of the experiment. For instance characterizing small RNAs requires different procedures than measuring changes in gene expression. Other variations are purely technical and have little or no impact on the final analysis.

In all cases an RNA-seq experiment involves making a collection of cDNA fragments which are flanked by specific constant sequences (known as adapters) that are necessary for sequencing (see figure 2). This collection (referred to as a library) is then sequenced using NGS which produces millions of short sequence reads that correspond to individual cDNA fragments.

A typical RNA-seq experiment consists of the following steps (depicted in figure 3):

  1. Isolate and purify the starting RNA.
  2. Convert it to cDNA.
  3. Convert the cDNA to a library ready for sequencing.
  4. Sequence using one of the available NGS platforms.
  5. Analyze the resulting short sequence-reads.

Although all RNA-seq experiments include these steps, there are many experimental details that must be considered before beginning an experiment.

Figure 2. RNA-seq is performed on short cDNAs derived from fragmented input RNA.
Figure 3. The four primary steps of an RNA-seq experiment. Note: Click on a box to view a detailed discussion of that step.