HomeCLC FAQ - Analyses-related questionsRNA-seqHow can I analyze and visualize strand specific RNA-Seq data?

8.5. How can I analyze and visualize strand specific RNA-Seq data?

If your RNA-Seq data is prepared using a strand specific protocol, you can analyze it by selecting either the strand specific option Forward or Reverse in the Mapping options step of the RNA-Seq Analysis wizard. If selecting the option Forward, the forward strand for each gene will be used as reference, whereas if selecting the option Reverse, the reverse strand for each gene will be used. The choice of option depends on whether you have used a forward or reverse strand protocol. The option Both will use both strands as reference and should be applied if you have non-strand specific RNA-Seq data.

The strand specific option only applies to the annotated gene regions. In the inter-genic regions both the Plus and Minus strand of the Reference Genome Track will be used as reference.

The results from the RNA-Seq analysis can be visualized in a Track List/Genome Browser view. Here, the reads are depicted against the Plus strand in the Reference Genome Track. Thus, if you have used the option Forward, the single or paired reads will be visualized map in their forward direction to genes on the Plus strand and in reverse complement direction to genes on the Minus strand. If the option Reverse was selected, this would be opposite.

When working with single reads, you can, by default, see the orientation of the reads in the Reads Track, as reads mapped in the forward direction are colored Green, while reads mapped in their reverse orientation are colored  Red. Likewise, you can by default see the orientation of paired in the Reads Track. Paired reads that mapped in their forward orientation will be shown in blue, while the paired reads that mapped in reverse orientation will be colored light blue.

Example screen shots with single and paired reads prepared with a strand specific protocol for the Forward strand can be found in the sections below.

 

Single reads:

 

Figure 1: Single reads mapped with the strand specific option Forward to a gene on the Plus strand. The reads are depicted in their forward orientation, which is visualized by the color green.

 

 

Figure 2: Single reads mapped with the strand specific option Forward to a gene on the Minus strand. The reads are depicted in their reverse orientation, which is visualized by the color red.

 

Figure 3: Single reads mapped with the strand specific option Forward to two overlapping genes on each strand. As a strand specific protocol has been used it is now possible to know from which gene the reads originate. The green are from the gene on the Plus strand and the red are from the gene on the Minus strand.

 

 

Paired reads:

 

Figure 4: Paired reads mapped with the strand specific option Forward to a gene on the Plus strand. The paired reads are depicted in their forward orientation, which is visualized by the color blue, also when the option Highlight reverse paired reads in the side panel of the Reads Track is checked.

 

Figure 5: Paired reads mapped with the strand specific option Forward to a gene on the Minus strand. The paired reads are depicted in their reverse orientation, which can be visualized by the color light blue, when the option Highlight reverse paired reads in the side panel of the Reads Track is selected.

 

Figure 6: Paired reads mapped with the strand specific option Forward to two overlapping genes on each strand. As a strand specific protocol has been used it is now possible to know from which gene the reads originate. The blue read are from the gene on the Plus strand and the light blue are from the gene on the Minus strand.

 

 

 

Background


If using a strand specific protocol for creating the RNA-seq library, then the strand information will be maintained.

This information can then be used to ensure that the reads map to the correct gene from which the originate. This is important in cases where you have overlapping genes located on different strands or paralogous genes located on different strands of the same chromosome. Without a strand specific protocol, this would not be possible. For more information on this please see Parkhomchuk et al., 2009.

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