Subsections


RNA-Seq result handling

Clicking Next will allow you to specify the output options as shown in figure 31.41.

Image mrna_seq_step4
Figure 31.44: 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 31.42).

Image mrna_seq_result_table
Figure 31.45: RNA-Seq results shown in a table view.

By creating a Track list, the graphical view can be shown together with the read mapping track and tracks from other samples:

        File | New | Track List (Image trackset_16_n_p)

Select the mapping and expression tracks of the samples you wish to visualize together and select the annotation tracks used as reference for the RNA-Seq and click Finish.

Once the track list is shown, double-click the label of the expression track to show it in a table view.

Clicking a row in the table makes the track list view jump to that location, allowing for quick inspection of interesting parts of the RNA-Seq read mapping (see an example in figure 31.43).

Image mrna_seq_result_contig
Figure 31.46: RNA-Seq results shown in a split view with an expression track at the bottom and a track list with read mappings of two samples at the top.

Reads spanning two exons are shown with a dashed line between each end as shown in figure 31.43, and the thin solid line represents the connection between two reads in a pair.

When doing comparative analysis, double click on one of the Expression or Statistical Comparison tracks in a track list to get its table view. Then click on the "Select genes in other views" button in any other table or expression browser will cause the track list to zoom to the selected gene.

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 31.44 for an example subset of an expression track).

Image mrna_seq_result
Figure 31.47: 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 31.35, 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 31.45 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 31.48: 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.