QIAGEN Bioinformatics Manuals

Align and count - QIAseq UPX 3' Transcriptome Kit

The analysis workflow is based on the Biomedical Genomics Analysis plugin template workflow QIAseq Quantify QIAseq UPX 3' described at:

It includes the steps below. Specific parameters are listed in Appendix C.

Demultiplex Reads. Reads are grouped into samples based on the sample-specific adapters.
Remove and Annotate with Unique Molecular Index. The UMI and common sequence prefixes are removed from the reads. Instead, a UMI annotation is added to each read to retain fragment identity.
Trim Reads. Short, internal polyA sequences are removed from R1 reads, and the short R2 reads are discarded.
Trim Reads. PolyA and polyG sequences are removed from R1.
Create UMI Reads from Reads. Sequencing reads are merged to UMI reads based on the unique molecular indexes (UMIs).
Trim Reads. Low quality nucleotides, ambiguous nucleotides, and adapter sequences are removed.
RNA-Seq Analysis. UMI reads are mapped to transcripts, and expression values for each gene are obtained by summing the transcript counts belonging to the gene.

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