QIAGEN Bioinformatics Manuals

Align and count - QIAseq UPXome RNA Lib Kit

RNA-seq Analysis Portal supports three cDNA synthesis protocols: N6-T RT primer, ODT-T RT primer, or combined N6-T RT and ODT-T RT primers. The analysis workflows are based on the corresponding Biomedical Genomics Analysis QIAseq template workflows described under
https://resources.qiagenbioinformatics.com/manuals/biomedicalgenomicsanalysis/2220/index.php?manual=UPXome_RNA.html

N6-T RT primer and ODT-T RT primer protocols

For the N6-T RT primer and ODT-T RT primer protocols, the analysis workflows include the following steps. Specific parameters are listed in Appendix C.

Demultiplex Reads. Reads are grouped into samples based on the sample-specific adapters.
Trim Reads. Low quality, ambiguous nucleotides and adapter sequences are removed.
RNA-Seq Analysis. Reads are mapped to transcripts, and expression values for each gene are obtained by summing the transcript counts belonging to the gene.

N6-T RT + ODT-T RT primer protocol

For the combined N6-T RT and ODT-T RT primer protocol, the analysis workflow includes two sections, A and B. The steps involved in each section are listed below. Specific parameters are listed in Appendix C.

Analysis workflow section A:

Demultiplex Reads. Reads are grouped into samples based on the sample-specific adapters.
Trim Reads. Adapter sequences are removed.

Analysis workflow section B:

Section B is split in two subsections, B1 and B2. B1 creates gene level expression results using all reads as input (N6-T and ODT-T reads combined). B2 generates transcript level expression values from N6-T reads only. Both subsections contain the following steps:

Trim Reads. Low quality, ambiguous nucleotides are removed.
RNA-Seq Analysis. Reads are mapped to transcripts. For the gene expression, expression values for each gene are obtained by summing the transcript counts belonging to the gene.

Results from the B1 subsection are used for subsequent differential gene expression analysis and as input for the Experiment summary and QC report Quality control tab. The Quality control tab is split into two sections and shows statistics for all reads in one section and the N6 reads in a separate section.

The tools are described in more detail in the QIAGEN CLC Genomics Workbench and Biomedical Genomics Analysis plugin manuals:

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Align and count - QIAseq UPXome RNA Lib Kit