The QC for RNAscan Panels tool can be found in the Toolbox here:
Tools | QIAseq Panel Expert Tools () | QIAseq RNAscan Panel Expert Tools () | QC for RNAscan Panels ()
Specify a RNA-seq read mapping as input (figure 6.21).
In the next dialog (figure 6.22), specify the mRNA track and the primer track that are saved in the CLC_References folder of the Navigation Area when downloading the QIAseq RNAscan Panels hg38 Reference Data Set. You can also set a maximal distance between a read and a primer start for them to be considered matching. It is set by default to 0, which means that a read will not be considered as starting in the primer unless it maps exactly to the start of the primer.
The tool outputs a primer track with annotated read coverage and a report that recapitulates QC data (figure 6.23). The primer track gives information about each primer, as well as their read coverage, whether they overlap with target or housekeeping genes.
A QC for RNAscan Panels Report contains the following information:
- Total number of mapped reads, counting paired reads as two.
- Total number of mapped reads(-pair)s, counting paired reads as one.
- Target genes: genes targeted by the primers.
- Number of target genes.
- Primers in target genes: how many primers are within the target genes regions.
- Mean read coverage per target gene primer (exact start position match): coverage from reads that start exactly at the primer start site.
- Fraction of mapped read(-pair)s exactly matching primers [%].
- Primers outside gene regions: gDNA control primers outside target gene regions, used for detection of DNA contamination of samples.
- Mean read coverage per non-gene primer: a number higher than 0 indicates DNA contamination of the sample. A mean gDNA coverage around 50 reads or higher may increase false positive signal level.
- DNA contamination [%]: calculated using the Mean read coverage per target gene primer and the Mean read coverage per non-gene primer metrics. In general, if the Mean read coverage per non-gene primer metric is around 50 or higher, the chances for a false positive may be higher.
- Primers in reference genes [COPA, MRPS14, CIAO1, UBE3C]: QIAseq RNAscan panels typically include four reference gene primers.
- Mean read coverage per reference gene control primer: the reference genes should have a mean coverage of at least 300 reads, otherwise the effective input is too low, and false negatives are expected.
- Target gene versus reference gene coverage ratio [%]