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• Introduction
• General Description
  • Supported sample kits and sequencing platforms
  • Accessing RNA-seq Analysis Portal
  • Analysis credits for RNA-seq Analysis Portal
• Getting Started
  • Upload sequencing data
  • Align and count
  • Create experiment
  • View and filter results
  • What's next - send results downstream
  • Design follow-up experiments in GeneGlobe
• User Interface
  • Analysis Portal page
    • Front page and Projects overview
    • Project view
    • Send copy of project
  • Samples page
• Starting Analyses
  • Align and count dialog
    • QIAseq UPX 3' Transcriptome Kit and QIAseq UPXome RNA Lib Kit specific settings
  • Create experiment dialog
    • Sample grouping
    • Experimental design
  • Compare analyses dialog
• Viewing Results
  • Experiment summary and QC report
  • Differential expression view
    • Feature table
    • Volcano plot
    • Heat map
    • Biological insights table
    • Filter
    • Send results to GeneGlobe
    • Send results to QIAGEN IPA
  • Comparison view
• RNA-seq Analysis Portal analysis explained
  • Align and count analysis workflows
    • Align and count - QIAseq miRNA Library Kit
    • Align and count - QIAseq UPX 3' Transcriptome Kit
    • Align and count - QIAseq UPXome RNA Lib Kit
    • Align and count - Demultiplexed RNA sample kits
  • Create experiment analysis workflow
  • Taxonomic profiling of unmapped reads
  • Compare analyses
• Appendix A - Sequence file naming pattern
• Appendix B - Reference and annotation information
  • General references and annotations
  • Sample kit specific resources
• Appendix C - Analysis workflow versions, species support and parameters
  • QIAseq miRNA Library Kit (Illumina)
  • QIAseq miRNA Library Kit (Thermo Fisher)
  • QIAseq UPX 3' Transcriptome Kit
  • QIAseq UPXome RNA Lib Kit
  • QIAseq UPXome RNA Lib Kit (N6-T RT primer)
  • QIAseq UPXome RNA Lib Kit (ODT-T RT primer)
  • QIAseq UPXome RNA Lib Kit (N6-T + ODT-T RT primers)
  • QIAseq FastSelect RNA Lib Kit (N6-T RT primer)
  • QIAseq FastSelect RNA Lib Kit (ODT-T RT primer)
  • QIAseq FastSelect RNA Lib Kit (N6-T + ODT-T RT primers)
  • QIAseq Stranded mRNA Select/Stranded Total RNA Lib Kit
  • TruSeq Stranded Total RNA Library Prep (Human/Rat, Gold, Globin)
  • Illumina Stranded Total RNA Prep with Ribo-Zero Plus
  • NEBNext Ultra II Directional RNA Library Prep Kit for Illumina
  • KAPA RNA HyperPrep Kit
  • SMARTer Stranded Total RNA Sample Prep Kit - HI Mammalian/Low Input Mammalian
  • Collibri Stranded RNA Library Prep Kit for Illumina Systems
• Appendix D - RNA-seq Analysis Portal for QIAGEN IPA users
• Appendix E - Quality control details
  • QIAseq miRNA Library Kits
  • QIAseq UPX 3' Transcriptome Kits
  • QIAseq Stranded RNA Library Kits (FastSelect RNA Library Kit, Stranded mRNA/Stranded Total RNA Library Kits), QIAseq UPXome Kit, and all third-party kits

Align and count - Demultiplexed RNA sample kits

This Align and count analysis workflow is used for the remaining sample kits. These do not leverage unique molecular indexes. Sample data must be demultiplexed prior to being uploaded to RNA-seq Analysis Portal.

Sample kits

• QIAseq Stranded mRNA Select/Stranded Total RNA Lib Kit (QIAGEN)
• QIAseq FastSelect RNA Lib Kit (QIAGEN)
• QIAseq UPX 3' Transcriptome Kit (with already-demultiplexed input data selected)
• QIAseq UPXome RNA Lib Kit (with already-demultiplexed input data selected)
• TruSeq Stranded Total RNA Library Prep (Human/Rat, Gold, Globin)(Illumina)
• Illumina Stranded Total RNA Prep with Ribo-Zero Plus (Illumina)
• NEBNext Ultra^TM II Directional RNA Library Prep Kit for Illumina (New England Biolabs)
• KAPA RNA HyperPrep Kit (Roche Sequencing solutions)
• SMARTer Stranded Total RNA Sample Prep Kit - HI Mammalian/Low Input Mammalian (Takara Bio)
• Collibri Stranded RNA Library Prep Kit for Illumina Systems (Thermo Fisher Scientific)

The analysis workflow includes the steps below. Parameters for the individual sample kits are listed in Appendix C.

• Trim Reads. Low quality and 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.

The tools are described in more detail in the QIAGEN CLC Genomics Workbench manual:

• https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/2103/index.php?manual=Trim_Reads.html
• https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/2103/index.php?manual=RNA_Seq_Analysis.html

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