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Interpreting the RNA-Seq analysis result

The main results of the RNA-Seq are two expression tracks: one summarizing expression at the gene level (called GE) and one summarizing expression at the transcript level (called TE). Note that the latter is only produced if the "Genome annotated with genes and transcripts" option is selected in figure 27.4.

Both tracks can be shown in a Table () and a Graphical () 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 ()

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 27.12).

Image mrna_seq_result_contig-web
Figure 27.12: 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 27.12, and the thin solid line represents the connection between two reads in a pair.

When doing comparative analysis and opening an experiment (see Experimental design) and a track list, clicking a row in the experiment will cause the track list to jump to the corresponding position, allowing for quick inspection of the reads underlying the counts in the experiment. Please note that at least one of the expression tracks used in the experiment have to be included in the track list in order for the link between the two to work.

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 holds information about counts and expression values for each gene. It can be opened in a Table view () allowing sorting and filtering on all the information in the track (see figure 27.13 for an example subset of an expression track).

Image mrna_seq_result
Figure 27.13: 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:

Name. This is the name of the gene or the reference sequence, if the One reference sequence per transcript is used.
Chromosome and region. The position of the gene on the genome.
Expression value. This is based on the expression measure chosen as described in Calculating expression values from RNA-Seq.
Gene length The length of the gene as annotated.
RPKM. This is the expression value measured in RPKM [Mortazavi et al., 2008]: $\emph{RPKM} = \frac{\emph{total exon reads}}{\emph{mapped reads(millions)} \times \emph{exon length (KB)}}$ . See exact definition below.
Unique gene reads. This is the number of reads that match uniquely to the gene or its transcripts.
Total gene reads. This is all the reads that are mapped to this gene -- both reads that map uniquely to the gene or its transcripts and reads that matched to more positions in the reference (but fewer than the 'Maximum number of hits for a read' parameter) which were assigned to this gene.
Transcripts annotated. The number of transcripts based on the mRNA annotations on the reference. Note that this is not based on the sequencing data - only on the annotations already on the reference sequence(s).
Detected transcripts. The number of annotated transcripts to which reads have been assigned (see the description of transcript-level expression below).
Exon length. The total length of all exons (not all transcripts).
Exons. The total number of exons across all transcripts.
Unique exon reads. The number of reads that match uniquely to the exons (including across exon-exon junctions).
Total exon reads. Number of reads mapped to this gene that fall entirely within an exon or in exon-exon or exon-intron junctions. As for the 'Total gene reads' this includes both uniquely mapped reads and reads with multiple matches that were assigned to an exon of this gene.
Ratio of unique to total (exon reads). The ratio of the unique reads to the total number of reads in the exons. This can be convenient for filtering the results to exclude the ones where you have low confidence because of a relatively high number of non-unique exon reads.
Unique exon-exon reads. Reads that uniquely match across an exon-exon junction of the gene (as specified in figure 27.12). The read is only counted once even though it covers several exons.
Total exon-exon reads. Reads that match across an exon-exon junction of the gene (as specified in figure 27.12). As for the 'Total gene reads' this includes both uniquely mapped reads and reads with multiple matches that were assigned to an exon-exon junction of this gene.
Unique intron reads. Reads that uniquely map partly or entirely within an intron.
Total intron reads. All reads that are mapped partly or entirely within to the introns of the gene.
Ratio of intron to total gene reads. This can be convenient to identify genes with poor or lacking transcript annotations. If one or more exons are missing from the annotations, there will be a relatively high number of reads mapping in the intron.

Transcript-level expression
If the "Genome annotated with genes and transcripts" option is selected in figure 27.4, a transcript-level expression track is also generated.

The track can be opened in a Table view () 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.

Name. This is the name of the transcript, if the One reference sequence per transcript is used.
Chromosome and region. The position of the gene on the genome.
Expression value. This is based on the expression measure chosen as described in Calculating expression values from RNA-Seq.
RPKM. This is the expression value measured in RPKM [Mortazavi et al., 2008]: $\emph{RPKM} = \frac{\emph{total exon reads}}{\emph{mapped reads(millions)} \times \emph{exon length (KB)}}$ . See exact definition below.
Relative RPKM. The RPKM for the transcript divided by the maximum of the RPKM values among all transcripts of the same gene. This value describes the relative expression of alternative transcripts for the gene.
Gene name. The name of the corresponding gene.
Transcript length. This is the length of the transcript.
Exons. The total number of exons in the transcript.
Transcript ID. The transcript ID is taken from the transcript_id note in the mRNA track annotations and can be used to differentiate between different transcripts of the same gene.
Unique exon reads. The number of reads that match uniquely to the exons (including across exon-exon junctions).
Total exon reads. The number of reads mapped to this gene that fall entirely within an exon or across an exon-exon junctions. As for the 'Total gene reads' this includes both uniquely mapped reads and reads with multiple matches that were assigned to an exon of this gene.
Ratio of unique to total (exon reads). The ratio of the unique reads to the total number of reads in the exons. This can be convenient for filtering the results to exclude the ones where you have low confidence because of a relatively high number of non-unique exon reads.
Unique exon-exon reads. Reads that uniquely match across an exon-exon junction of the gene (as specified in figure 27.12). The read is only counted once even though it covers several exons.
Total exon-exon reads. Reads that match across an exon-exon junction of the gene (as specified in figure 27.12). As for the 'Total gene reads' this includes both uniquely mapped reads and reads with multiple matches that were assigned to an exon-exon junction of this gene.

Definition of RPKM
RPKM, Reads Per Kilobase of exon model per Million mapped reads, is defined in this way [Mortazavi et al., 2008]:

$\displaystyle \emph{RPKM} = \frac{\emph{total exon reads}}{\emph{mapped reads(millions)} \times \emph{exon length (KB)}}.$

Total exon reads: This value can be found in the column with header Total exon reads in the expression track. This is the number of reads that have been mapped to exons (either within an exon or at the exon junction). When the reference genome is annotated with gene and transcript annotations, the mRNA track defines the exons, and the total exon reads are the reads mapped to all transcripts for that gene. When only genes are used, each gene in the gene track is considered an exon. When an un-annotated sequence list is used, each sequence is considered an exon.
Exon length: This is the number in the column with the header Exon length in the expression track, divided by 1000. This is calculated as the sum of the lengths of all exons (see definition of exon above). Each exon is included only once in this sum, even if it is present in more annotated transcripts for the gene. Partly overlapping exons will count with their full length, even though they share the same region.
Mapped reads: The sum of all mapped reads as listed in the RNA-Seq analysis report. Please note that the option to Map to gene regions only will affect the number of mapped reads, since all intergenic reads will not be mapped if this option is selected. This means that comparison of RPKM values between samples should only be carried out if this parameter was set in the same way for all samples.

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Interpreting the RNA-Seq analysis result