Demultiplex QIAseq UPX 3' reads

The UPX 3' RNA application of the Analyze QIAseq Panels guide offers a Demultiplex tool to be run before starting the Quantify QIAseq UPX 3' workflow. The workflow requires the reads to be sequenced with a QIAseq UPX 3' Transcriptome Kit or a QIAseq UPX 3' Targeted RNA Panel.

Click Run to open the tool's wizard. In the first dialog, select the reads you want to demultiplex, and click Next. In the following dialog (figure 9.1), how the reads were generated - either NextSeq or MiSeq/HiSeq - and the appropriate plate size used for sequencing are automatically inferred from the 10,000 first reads in the sample, but can be manually specified as well. Note: with data from NovaSeq, select the MiSeq/HiSeq option.

It is also possible to enable the option that will allow one mismatch per barcode.

Image upxdemultiplex
Figure 9.1: The demultiplex tool offers to visualize which wells were used during sequencing, and which should be selected for demultiplexing.

The wells used in an experimental setup and identified in the plate mock up by a blue dot. To be demultiplexed, each well should be selected:

Once they are selected, wells are highlighted with a dark blue circle around the well. The button Invert select all wells that were previously deselected.

The tool will output a sequence list for each well selected in the previous step. In addition, you can choose to Create a report and a list of reads for which no barcodes were found. The report indicates how many reads were found for each barcode identified, presented in a table and as a graph.