RNA-Seq report

An example of an RNA-seq report generated if you choose the Create report option is shown in figure 28.16.

Image mrna_seq_report
Figure 28.16: Report of an RNA-Seq run.

The report is a collection of the sections described below, some sections included only based on the input provided when starting the tool. If a section is flagged with a pink highlight, it means that something has almost certainly gone wrong in the sample preparation or analysis. A warning message tailored to the highlighted section is added to the report to help troubleshoot the issue. The report can be exported in PDF or Excel format.

Selected input sequences

Information about the sequence reads provided as input, including the number of reads in each sample, as well as information about the reference sequences used and their lengths.


Information about the total number of genes and transcripts found in the reference:

Spike-in quality control

Read quality control

This section includes:

Mapping statistics

Shows statistics on:

Fragment statistics

Distribution of biotypes

Table generated from biotype annotations present on the input gene or mRNA tracks. If using both gene and mRNA tracks, the biotypes in the report are taken from the mRNA track.

The biotypes are "as a percentage of all transcripts" or "as a percentage of all genes". For a poly-A enrichment experiment, it is expected that the majority of reads correspond to protein-coding regions. For an rRNA depletion protocol, a variety of non-coding RNA regions may also be observed. The percentage of reads mapping to rRNA should usually be <15%.

If over 15% of the reads mapped to rRNA, it could be that the poly-A enrichment/rRNA depletion protocol failed. The sample can still be used for differential expression and variant calling, but expression values such as TPM and RPKM may not be comparable to those of other samples. To troubleshoot the issues in future experiments, check for rRNA depletion prior to library preparation. Also, if an rRNA depletion kit was used, check that the kit matches the species being studied.

Gene/transcript length coverage

Plot showing the normalized coverage across a gene/transcript body for four different groupings of gene/transcript length (figure 28.19).

Image lengthcoverage
Figure 28.19: Gene/transcript length coverage plot.

To generate this plot, every transcript is rescaled to have a length of 100. For every read that is assigned to a transcript, we get its start and end coordinates in this "transcript-length-normalized" coordinate system [0,100]. We then increment counters from the read start position to the read end position. After all the reads have been counted, the average 5' count is the average value of the counters at position 0,1,2...49. The average 3' count is the value at positions 51,52,53...100. The difference between average 3' and 5' normalized counts is the difference between these values as a percentage of the maximum number of counts seen at any position.

The lines should be flat in the center of the plot, and the plot should be approximately symmetric. An erratic line may indicate that there are few genes/transcripts in the given length range. Lines showing normalized count higher on the 3'end indicates the presence of polyA tails in the reads, consequence of degraded RNAs. Future experiments may benefit from using an rRNA depletion protocol.

In the table below the plot, a difference between average 3' and 5' normalized counts higher than 25 warns that variants may not be called in low coverage regions, and that TPM or RPKM values may be unreliable. Most transcripts are <10000 bp long, so a warning is raised if many reads map to features longer than this. One possible cause is that no mRNA track has been provided for an organism with extensive splicing.