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 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 31.33.
This is a simple gene with three exons and two splice variants. The transcripts are extracted as shown in figure 31.34.
Next, the reads are mapped against all the transcripts, and to the whole genome. For more information about the read mapper, see Map Reads to Reference.
From this mapping, the reads are categorized and assigned to the transcripts using the EM estimation algorithm, and expression values for each gene are obtained by summing the transcript counts belonging to the gene.
- Reads and reference settings
- Mapping settings
- The EM estimation algorithm
- Expression settings
- RNA-Seq result handling
- RNA-Seq report
- Gene fusion reporting