RNA-Seq analysis

Two tools are available for RNA-seq analysis, the tool RNA-Seq Analysis and the tool Create Fold Change Track Based on an annotated reference genome, the CLC Cancer Research Workbench supports RNA-Seq analysis by mapping next-generation sequencing reads and counting and distributing the reads across genes and transcripts. Subsequently, the results can be used for expression analysis using the tools in the Transcriptomics Analysis toolbox.

The tool for RNA-Seq analysis can be found here:

        Toolbox | Transcriptomics Analysis (Image transcriptomics_closed_16_n_p) | RNA-Seq Analysis (Image rna_seq_group_closed_16_n_p)

The approach taken by the CLC Cancer Research Workbench is based on [Mortazavi et al., 2008].

The following describes the overall process of the RNA-Seq analysis when using an annotated eukaryote genome. See Specifying reads, reference genome and mapping settings for more information on other types of reference data.

The RNA-Seq analysis is done in several steps: First, all genes are extracted from the reference genome (using a gene track). Next, all annotated transcripts are extracted (using an mRNA track). If there are several annotated splice variants, they are all extracted.

An example is shown in figure 28.1.

Image rnaseq_explained1
Figure 28.1: A simple gene with three exons and two splice variants.

This is a simple gene with three exons and two splice variants. The transcripts are extracted as shown in figure 28.2.

Image rnaseq_explained2
Figure 28.2: All the exon-exon junctions are joined in the extracted transcript.

Next, the reads are mapped against all the transcripts plus the entire gene (see figure 28.3) and optionally to the whole genome.

Image rnaseq_explained3
Figure 28.3: The reference for mapping: all the exon-exon junctions and the gene.

From this mapping, the reads are categorized and assigned to the genes (elaborated later in this section), and expression values for each gene and each transcript are calculated.

Details on the process are elaborated in the following sections, which describe how to run RNA-seq analyses.