Align and count - QIAseq miRNA Library Kit
The analysis workflow is based on the Biomedical Genomics Analysis plugin template workflow QIAseq miRNA Quantification described at:
It includes the steps below. Specific parameters are listed in Appendix C.
- Create UMI Reads for miRNA. Sequencing reads are merged to UMI reads based on the unique molecular indexes (UMIs).
- Quantify miRNA. miRNAs are counted and annotated using miRBase and custom databases. Expression levels are generated for the mature miRNAs. For species that support piRNA analysis (see Appendix B), unmapped reads are aligned to a piRNA database to generate piRNA counts.
- RNA-Seq Analysis. Unmapped reads from the Quantify miRNA step are aligned to transcript sequences. Gene-level expression values are then calculated by summing the counts of all transcripts associated with each gene.
The tools are described in more detail in the QIAGEN CLC Genomics Workbench and Biomedical Genomics Analysis plugin manuals:
- https://resources.qiagenbioinformatics.com/manuals/biomedicalgenomicsanalysis/2510/index.php?manual=Create_UMI_Reads_miRNA.html
- https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/2502/index.php?manual=Quantify_miRNA.html
- https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/2502/index.php?manual=RNA_Seq_Analysis.html