Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus FAQs

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  • General

  • The Ribo-Zero Plus rRNA removal mix depletes globin mRNAs from human, mouse and rat blood samples. Use of this kit with other organisms has not been tested and is not supported.

    The new Ribo-Zero Plus leverages a different depletion mechanism compared to legacy Ribo-Zero. Ribo-Zero Plus uses DNA probes and enzymatic depletion to remove abundant transcripts from total RNA samples. Legacy Ribo-Zero leverages biotinylated RNA probes to pull down abundant transcripts via streptavidin beads. When paired with our new Illumina Stranded Total RNA prep, Ribo-Zero Plus offers key advantages that enable enhanced study flexibility. These advantages include a “one tube (single kit) to deplete multiple species” functionality, enhanced depletion at lower total RNA inputs, and a more precise mechanism for depletion of targeted transcripts.

    While our long-term goal is to have a design tool available for customers to design their own Ribo-Zero Plus probes, we do not have a tool available and unfortunately cannot confirm a timeline for such a tool.

    Illumina has tested and demonstrated effective rRNA depletion with the new Ribo-Zero Plus kit of E. coli (gram-negative) and B. subtilis (gram-positive) bacteria samples only. rRNA depletion of other bacterial samples has not been tested and performance may vary.  While the new Ribo-Zero Plus targets the same bacterial transcript regions as legacy Ribo-Zero bacteria, Ribo-Zero Plus leverages a more precise targeting mechanism than Ribo-Zero legacy. Because of this, Ribo-Zero Plus may sometimes be less tolerant to bacterial species that are more highly divergent in sequence from E. coli and B. subtilis.

    No, there is no Ribo-Zero Plus module for rRNA depletion of plant species for the new Illumina Stranded Total RNA Prep. Customers who need a plant depletion should use legacy TruSeq kits. 

    Ribo-Zero Plus depletes human globin transcripts and rRNAs from multiple species including human, mouse, rat, and bacteria (E. coli and B. subtilis). Illumina cannot guarantee rRNA removal of species outside of the following.

    Depletion target rRNAs targeted
    Human Cytoplasmic rRNAs 28S, 18S, 5.8S, 5S
    Human Mitochondrial rRNAs 12S, 16S
    Human Beta Globin transcripts HBA1, HBA2, HBB, HBG1, HBG2
    Mouse and Rat rRNA 16S, 28S
    Gram(-) Bacterial rRNAs E. coli: 5S, 16S, 23S
    Gram(+) Bacterial rRNAs B. subtilis: 5S, 16S, 23S
  • Analysis

    • DRAGEN RNA Pipeline- Performs fusion analysis and RNA quantification.
    • RNA-Seq Alignment- Aligns RNA-seq reads. Quantifies gene expression, calls small variants and gene fusions. Input for differential expression app.
    • Cufflinks Assembly & DE-Quickly assess novel transcript isoforms and gene expression levels from RNA-Seq Alignment results.