Output from the Quantify QIAseq RNA Expression workflow
The Quantify QIAseq RNA Expression template workflow produces expression tracks (
) for each sample together with a combined report containing QC metrics (
). These reports can be used as input to Combine Reports to make a summary report for many samples. A Workflow Result Metadata table keeping track of all generated output and a log file will also be generated as default outputs. See (figure 10.5).
Figure 10.5: The output files of the Quantify QIAseq RNA Expression workflow.
Expression tracks are displayed as tables listing genes included in the panel and several measures of their expression levels.
- Expression value The number of distinct UMIs seen for this gene.
- TPM (Transcripts per Million) The number of transcripts per million that come from this gene. This is computed as the relative abundance per million
.
- RPKM (Reads Per Kilobase per Million reads) There is no good definition of RPKM for targeted amplicon data. We therefore define RPKM to be equal to TPM, which preserves the expected property that RPKM is proportional to TPM.
- Total gene reads The number of distinct UMIs seen for this gene.
- Read counts The total number of reads mapping to this gene. Several reads may have the same UMI.
- UMI counts The number of distinct UMIs seen for this gene.
- Mean reads per UMI The mean number of reads for each UMI seen for this target.
Expression tracks be further analyzed using statistical tools from the RNA-Seq Analysis folder of the workbench, such as PCA for RNA-Seq and Create Heat Map for RNA-Seq.
QC metrics can be found in the combined report. They indicate how many reads were ignored and the reason they were not included in a UMI read. The "Accepted reads" column contains the numbers of reads that passed the filtering carried out by Quantify QIAseq RNA, as described in Targeted RNA tools detailed description.
When you are designing a customized panel online, you will see that there is an option to add a set of 6 "GDC controls". These values are your negative controls of genomic DNA. You can also see these in the target regions where they have names like "GDC_CONTROL_06". The number of control targets with more than 10 UMI reads are listed in the "Expressed GDC controls" table cell. This cell will be colored pink and a warning will be shown if any controls are expressed.
Why does the workflow not produce a read mapping?
The very short target regions in a QIAseq Targeted RNA Panel are not suited to downstream analyses that require a read mapping, such as variant calling. If a read mapping is desired, for example to investigate suspected off-target effects, we recommend using the Map Reads to Reference tool.