Quantify QIAseq RNA
The Quantify QIAseq RNA tool can be found in the Toolbox here:
Toolbox | Biomedical Genomics Analysis (
) | QIAseq Tools (
) | Quantify QIAseq RNA (
)
The tool uses RNA-Seq reads as input, together with a reference sequence and target regions that are saved in the CLC_References folder of the Navigation Area when downloading the QIAseq RNA Panels hg38 reference data set.
We recommend using reads that have already been trimmed, as done for example in the Quantify QIAseq RNA Expression template workflow, see Quantify QIAseq RNA Expression. Trimming increases the results accuracy.
The tool performs multiple steps, as described below.
Mapping
The reads are mapped to the target regions using Map Reads to Reference (see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Map_Reads_Reference.html) with default settings. "Auto-detect paired distances" is however disabled, as the automatic estimation of paired distances is not appropriate for targeted data.
The reference sequences of the target regions are created as follows:
- The sequence of the target regions is determined from the reference sequence.
- The exons of multi-exon targets are concatenated into one single target sequence.
- 12 ambiguous "N" nucleotides are prepended to the 5' region to account for UMIs.
Note that all other regions of the genome are ignored.
Filtering
Reads that successfully mapped to the target regions are removed if the UMI does not have the expected length of 12.
Merging
PCR and sequencing errors also happen in the UMIs. To account for this, UMIs can be merged as described in [Peng et al., 2015]. Briefly, UMIs that have a count of at most 2 or lower than 5% of the maximum observed UMI count, are considered for merging. For a UMI to be merged into another UMI, it must differ by at most one base pair and have a count that is at least 6 fold smaller.