Subsections


RNA-Seq result handling

Clicking Next will allow you to specify the output options as shown in figure 33.40.

Image mrna_seq_step4
Figure 33.40: Selecting the output of the RNA-Seq analysis.

The main results of the RNA-Seq analysis are Expression Tracks. A track with a name ending with (GE) summarizes expression at the gene level. If the "Genome annotated with genes and transcripts" option was selected, a second track, with a name ending in (TE), is produced, which summarizes expression at the transcript level.

In addition, other results can be generated using the following options:

Expression tracks

Both tracks can be shown in a Table (Image table) and a Graphical (Image annotation_track_16_n_p) view.

The expression track table view has the following options (figure 33.41).

Image mrna_seq_result_table
Figure 33.41: RNA-Seq results shown in a table view.

An example of a track list containing expression results is shown in figure 33.42. In that figure, one of the tracks referred to in the track list has been opened in a linked view. Clicking on a row in that table moves the focus in the track list to the location referred to in that row. General information about linked views is available in Linked views. Information on creating and working with track lists is provided in Track lists.

Image mrna_seq_result_contig
Figure 33.42: A track list containing RNA-Seq results is open in the top of the viewing area, with an expression track open in a linked view beneath.

Reads spanning two exons are shown with a dashed line between each end (figure 33.42). The thin solid line represents the connection between two reads in a pair.

Table views of some track types offer buttons at the bottom related to linked viewing. For example, Expression or Statistical Comparison tracks have buttons for putting the focus on selected genes or transcripts in other views.

Expression tracks can also be used to annotate variants using the Annotate with Overlap Information tool. Select the variant track as input and annotate with the expression track. For variants inside genes or transcripts, information will be added about expression (counts, expression value etc) from the gene or transcript in the expression track. Read more about the annotation tool in Annotate with Overlap Information.

Gene-level expression

The gene-level expression track (GE) holds information about counts and expression values for each gene. It can be opened in a Table view (Image table) allowing sorting and filtering on all the information in the track (see figure 33.43 for an example subset of an expression track).

Image mrna_seq_result
Figure 33.43: A subset of a result of an RNA-Seq analysis on the gene level. Not all columns are shown in this figure

Each row in the table corresponds to a gene (or reference sequence, if the One reference sequence per transcript option was used). The corresponding counts and other information is shown for each gene:

Transcript-level expression

If the "Genome annotated with genes and transcripts" option is selected in figure 33.35, a transcript-level expression track (TE) is also generated.

The track can be opened in a Table view (Image table) allowing sorting and filtering on all the information in the track. Each row in the table corresponds to an mRNA annotation in the mRNA track used as reference.

Additional information to GE or TE tracks

Both GE and TE tables can offer additional information such as hyperlinks to various databases (e.g., ENSEMBL, HGNC (HUGO Gene Nomenclature Committee), RefSeq, GeneID, etc.) In cases where the mRNA track or the gene track provided have biotype information, a biotype column will be added to the table.

RNA-Seq reads track

A track containing the mapped reads can be generated by the tool if the option to do so is enabled. Details about viewing and editing of reads tracks are described in Tracks and Reads tracks.

If you have chosen the strand specific option when setting up your analysis, it may be helpful to note that the colors of mapped single reads represent the orientation of the read relative to the reference provided. When a gene track is provided along with the reference genome, the reads will be mapped using the strand you specified, but the coloring of the read will be relative to the reference gene. If the reads matches the orientation of the gene it is colored green, and if it is opposite to the orientation of the gene it is colored red. A summary list of the colors to expect with different combinations of gene orientation and strand specific mapping options is:

See figure 33.44 for an example of forward and reverse reads mapped to a gene on the plus strand.

Note: Reads mapping to intergenic regions will not be mapped in a strand specific way.

Although paired reads are colored blue, they can be viewed as red and green 'single' reads by selecting the Show strands of paired reads box, within the Read Mapping Settings bar on the right-hand side of the track.

Image strandspecificrna
Figure 33.44: A track list showing a gene and transcript on the plus strand, and various mapping results. The first reads track shows a mapping of two reads (one 'forward' and one 'reverse') using strand specific 'both' option. Both reads map successfully; the forward read colored green (because it matches the direction of the gene), and the reverse read colored red. The second reads track shows a mapping of the same reads using strand specific 'forward' option. The reverse read does not map because it is not in the correct direction, therefore only the green forward read is shown. The final reads track shows a mapping of the same reads again but using strand specific 'reverse' option. This time, the green forward read does not map because it is in the wrong direction, and only the red reverse read is shown.