General FAQ for Illumina Rapid RNA kits
What is new about the Illumina Rapid RNA prep technology?
Illumina Rapid RNA Prep technology introduces a modernized RNA‑seq library preparation workflow that is fast, simple, and flexible.
What's new
Proprietary chemistry that allows cDNA synthesis and adapter tagging via on-bead tagmentation in a single step, eliminating multiple enzymatic reactions and cleanups.
A streamlined, same‑day workflow reduces total library preparation and enrichment time to less than one working day.
The chemistry is optimized for low‑input and degraded RNA, including challenging sample types such as FFPE.
Support for high‑plex Unique Dual Indexing (UDIs) enables safe, large‑scale multiplexing on patterned flow cells.
A modular design allows the same core library prep to be paired with multiple enrichment strategies, including RNA exome, targeted panels, and custom content.
Do Illumina Rapid RNA products have their own, dedicated user guides and product pages?
Yes, Illumina Rapid RNA products have their own dedicated Product Documentation as well as product pages. The complete list of Rapid products is as follows.
Product Pages:
Product Support pages:
What product contents and/or workflow changes achieve these differences?
The speed gains of Illumina Rapid RNA Prep are driven by a one-step prep reaction that combines first and second strand cDNA synthesis with EBLT(S) tagmentation beads. The duration of some thermal cycler steps are also reduced.
What are the key differences users should consider when running Rapid RNA versus other RNA prep methods?
Rapid RNA prep workflows produce non-stranded data with reads aligned to both the forward and reverse strands of the cDNA. The DRAGEN RNA App can analyze both Stranded and non-stranded RNA libraries, defined in the Library type under Advanced Settings. The default setting of Autodetect will correctly analyze Rapid RNA prep as non-stranded.
Note that Illumina Stranded mRNA and Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus library prep kits produce stranded data. The earlier-generation Illumina RNA Prep with Enrichment (IRPE) kit and other preps that use the same chemistry (eg, Viral Surveillance Panel v2 (VSPv2), Respiratory Pathogen ID/AMR Enrichment Panel Kit (RPIP), and Respiratory Virus Enrichment Kit (RVEK) which utilizes the Respiratory Virus Oligo Panel v2 (RVOP v2)) have always used tagmentation and generated non-stranded data as well.
Which Exome panel is included in the Rapid RNA Prep with Exome 2.5 Enrichment?
The Illumina Rapid RNA Prep with Exome 2.5 Enrichment (Rapid Exome 2.5) includes the box for Twist Bioscience for Illumina Exome 2.5 Panel 20076914, which is identical to the panel available in the Illumina DNA Prep with Exome 2.5 Enrichment v2, (S) Tagmentation Set B (96 Samples, 12-plex) 20155541 and Illumina DNA Prep with Exome 2.5 Enrichment, (S) Tagmentation Set B (96 Samples, 12-plex) 20077595.
What is the difference between Ribo-Zero Plus and Ribo-Zero Plus Microbiome versions?
Ribo-Zero Plus Microbiome contains one additional tube of rRNA depletion probes, designed to target species found in the microbiome of human adult and infant stool samples.
What are the expected insert sizes and duplicate rates for Rapid workflows?
Rapid RNA produces smaller library size fragments of 120-170 bp on average, which is shorter than the TruSeq or Illumina Stranded workflows. There are higher duplicate rates with increase of 30-50% duplicate percentages compared to the Illumina Stranded RNA preps, due to the EBLT(S) based tagmentation. Overall duplicate rates are still low for >10ng input.
Rapid mRNA tagmentation shows slightly lower coverage at ends of transcripts. Slightly higher Coefficient of Variance (CV) of coverage with higher coverage in middle of transcripts. See figure below.
Is there any demo data on BaseSpace for Illumina Rapid RNA Preps?
Yes, Agilent UHR for mRNA and Total RNA, Brain Reference for Total RNA, SeraCare Fusion reference for Total RNA and Rapid RNA Exome 2.5 will be available in BaseSpace Demo data by July 2026.
**Can users use the RiboZero Plus with Rapid RNA Prep and Fast Hyb enrichment all in one workflow for Globin RNA depletion from blood samples?**Yes, all three components can be used in combination for Rapid RNA workflows, but Illumina has not tested this combination on blood samples for globin RNA depletion prior to Rapid RNA Prep followed by Exome or Custom Enrichment.
For any feedback or questions regarding this article (Illumina Knowledge Article #10209), contact Illumina Technical Support techsupport@illumina.com.
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