The Quantify QIAseq RNA tool is available as a stand-alone tool in the Toolbox.
Tools | QIAseq Panel Expert Tools () | QIAseq RNA Panel Expert Tools () | Quantify QIAseq RNA ()
It is there solely for the purpose of building additional workflows and we do not recommend to use the tool on its own as the trimming step included in the Quantify QIAseq RNA Expression ready-to-use workflow increases the accuracy of the results generated.
The tool works in a three-step process: 1) Mapping, 2) Filtering of mapped reads, 3) Merging of reads with similar UMIs.
RNA-Seq reads are mapped to the target regions. All other regions of the genome are ignored.
The reference sequences of the target regions are created as follows:
- The sequence of the target regions is determined from the BED file.
- The exons of multi-exon targets are concatenated into one single target sequence.
- 12 ambiguous "N" nucleotides are prepended to the 5' region to allow reads with UMIs to map without mismatch penalties.
Reads mapping to the targets are checked to ensure that the UMI has the expected length. Reads with UMIs that are as little as one base pair too long or too short are filtered away.
To be considered for a merge, a UMI has to have a count below 5% of the maximum count.
To be merged, the count difference between the UMIs must be at least 6 fold, and they must differ from from another UMI by at most one base pair. The algorithm is described in more detail in [Peng et al., 2015].