Experiment summary and QC report
On the experiment analyses overview, the Experiment summary and QC tile is indicated by the PCA plot image (figure 40).
Figure 40: The Experiment summary and QC analysis tile includes a picture of a PCA plot.
Click on the tile to open it.
The Experiment summary and QC analysis result is a report divided into three tabs, Experiment summary, Samples, and Quality control.
Print or download report. To print or download the report, click on Print view above the three tabs (figure 41). Using your browser's print functionality, you can select a printer, or choose to save to PDF.
Experiment summary
The Experiment summary tab lists key information about the experimental design, including the attribute on which the experiment is based, and underlying samples (figure 41).
Figure 41: The first tab on the Experiment summary and QC report contains the Experiment summary.
Samples
The Samples tab contains a list of the samples included in the experiment and the specified sample attributes. Alongside this is a Principal Component Analysis (PCA) plot, colored by the attribute on which the experiment is based, indicated above the list (figure 42).
The PCA plot can help with identifying outlying samples for quality control purposes, and can provide you with a feel for the principal causes of variation in the dataset. If you have outlier samples, you may want to run the experiment again without these. If the Quality control tab metrics for outlier samples are skewed, this is additional evidence that you should consider removing them from your experiment.
Figure 42: The Samples tab lists samples and their attributes, and allows you to download expression values.
Download PCA plot. To download the PCA plot, hover the plot and click on PCA plot. In the dialog, select image resolution, and click on Download (figure 43).
Figure 43: The Download PCA plot dialog with options for image resolution.
Download expression values. To download sample expression values, click on Expression values above the table and select the expression values you wish to download. The available expression values differ between sample kits.
miRNA sample kit expression values:
- Mature miRNA values: Name, count, CPM (Counts per million, TMM normalized)
- IsomiR counts: Name, sequence, count, ambiguity tag (boolean)
- miRNA and piRNA counts: Counts for all samples in a single file
RNA sample kit expression values:
- Gene values - Total exon reads, RPKM (Reads Per Kilobase of exon model per Million mapped reads), TPM (Transcripts per million), CPM (Counts per million, TMM normalized)
- Transcript values - Total transcript reads, RPKM (Reads Per Kilobase of exon model per Million mapped reads), TPM (Transcripts per million), CPM (Counts per million, TMM normalized). Transcript values are not supported for 3' protocols such as QIAseq UPX 3' Transcriptome Kit.
RNA sample kit expression value downloads will contain all genes/transcripts of the reference genome, i.e. also biotypes not included in the differential expression analysis. A biotype column specifies the biotype of each feature.
CPM, RPKM, and TPM measures and the tool used to generate IsomiR counts are described in more detail in the QIAGEN CLC Genomics Workbench and Biomedical Genomics Analysis plugin manuals:
- https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/2103/index.php?manual=_expression_browser.html
- https://resources.qiagenbioinformatics.com/manuals//clcgenomicsworkbench/2103/index.php?manual=Extract_IsomiR_Counts.html
- https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/2103/index.php?manual=Expression_settings.html
This functionality is not available for samples created with analysis workflows introduced in RNA-seq Analysis Portal 1.1 or before, see the section Analysis worfklow versions in RNA-seq Analysis Portal.
Quality control
The Quality control tab displays several tables with sample quality control metrics from the individual Align and count analysis steps. The available metrics differ between sample kits.
The top table, Quality control summary, provides an overview of selected metrics like the number of reads, average quality scores, and mapped reads.
QIAseq miRNA Library Kit
The Quality control tab contains the two related tables Unique search sequences and Reads. Unique search sequences are groups of identical reads. The ratio between corresponding Unique search sequences values and Reads values indicates the redundancy of the particular biotype. In general, a higher level of redundancy is expected for miRNAs than for other biotypes. High redundancy for other biotypes may be indicative of issues with sequencing samples.
RNA sample kits
The Quality control summary metrics Mapped to total rRNA represents the sum of the corresponding cytoplasmic rRNA and mitochondrial rRNA (Mt_rRNA) values and indicates the level of rRNA contamination. It can be used as a measure of whether the rRNA depletion protocol worked as expected.
The Biotype distribution chart and table give insight into where reads mapped (figure 44). For a poly-A enrichment experiment, you expect that the majority of reads correspond to "protein_coding" regions. For other experiment types, a variety of non-coding RNA regions may also be observed. For an rRNA depletion protocol, the percentage of reads mapping to rRNA and Mt_rRNA combined should usually be <15%.
To download the biotype chart, hover the chart and click on Biotype chart.
Figure 44: The biotype chart shows the origin of the mapped reads. The large proportion of Mt_rRNA reads for three of the samples is an indication that no rRNA depletion protocol was used, or that if one was used, it did not work as expected.
The sections Taxonomic profile of unmapped reads and Taxonomic profiling summary will hold taxonomic profiling results for samples with more than 100,000 reads and where less than 75% of the reads could be mapped. A high level of unmapped reads could indicate contamination. The taxonomic profiling of the unmapped reads detects if those unmapped reads may originate from bacteria or archaea, and if so from which phyla. The plot (figure 45)and table under Taxonomic profile of unmapped reads show the relative abundance at phylum level.
To download the taxonomic profile chart, hover the chart and click on Taxonomic profile chart chart.
Figure 45: The taxonomic profile chart shows the relative abundance of phyla in unmapped reads of samples where less than 75% of the reads could be mapped.