RNA Library Preparation

RNA sequencing has revolutionized gene expression studies and transcriptome profiling. However, it has become apparent as this field continues to expand, that the quality of sequencing data is directly correlated to the quality of library preparation. To address this growing need, Kapa Biosystems offers a portfolio of products for the preparation of high-quality RNA libraries for Illumina® platforms. Kapa’s RNA library preparation kits include KAPA HiFi HotStart ReadyMix, which imparts low GC bias resulting in more even sequence coverage to maximize sequencing results. KAPA HiFi also allows for the increased detection of low-abundance transcripts. Additionally, all RNA-seq kits are designed to be automation-friendly on major liquid handlers.

NEW! KAPA Single-Indexed Adapter Kits are now available. For more information on these kits, visit the product page for your KAPA DNA or RNA library preparation kit.

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For Research Use Only. Not for use in diagnostic procedures.

Upfront RNA Enrichment Selection

A majority of total RNA consists of ribosomal RNA (rRNA), which is generally of little biological interest and can result in significant underutilization of sequencing capacity if not removed from an RNA sample. Selecting the appropriate RNA enrichment module to remove rRNA is critical, as there is a direct impact on resulting sequence data:

  • mRNA capture, where polyadenylated transcripts are enriched for over other species, such as rRNA, is only appropriate for fully intact samples and will focus sequencing on the protein-coding regions of the transcriptome
  • rRNA depletion, where rRNA transcripts are specifically targeted and depleted, can be used for degraded samples, including FFPE, and preserves other species of biological interest, including mRNA and noncoding RNAs

The two figures below visually illustrate some of the resulting data differences:

Sequencing-Data-Comparison

Sequencing data comparison of mRNA capture, KAPA RiboErase, and a total RNA sample having undergone the bioinformatic removal of rRNA reads. Both RNA enrichment methods effectively reduce rRNA carryover into the final library. However, mRNA capture biases the final read distribution towards exonic loci, while KAPA RiboErase preserves the intergenic/intronic/exonic read distribution observed in the bioinformatically-depleted sample. Libraries were constructed using 100 ng of Universal Human Reference. Data on file.


Sequencing data comparison of mRNA capture and KAPA RiboErase. In this example, KAPA RiboErase detects the non-coding RNA SCARNA12, outlined in red, while mRNA capture only detects the surrounding protein-coding transcripts. Libraries were constructed using 100 ng of Universal Human Reference. Data on file.

Sequencing data comparison of mRNA capture and KAPA RiboErase. In this example, KAPA RiboErase detects the non-coding RNA SCARNA12, outlined in red, while mRNA capture only detects the surrounding protein-coding transcripts. Libraries were constructed using 100 ng of Universal Human Reference. Data on file.

cDNA Library Prep Workflow Selection

Kapa provides two RNA-Seq library preparation workflow options. The KAPA Stranded RNA-Seq Library Preparation Kits offer a traditional workflow, while the KAPA RNA Hyper Prep product portfolio provides a highly-streamlined option, reducing the core library construction process by approximately 2 hours, or 30%. The following options are available for RNA library preparation:

No RNA Enrichment mRNA Capture Ribosomal Depletion
cDNA Library Prep Workflow Standard Streamlined Standard Streamlined Standard Streamlined
KAPA Stranded RNA-Seq Kits KAPA RNA HyperPrep Kits KAPA Stranded mRNA-Seq Kits KAPA mRNA HyperPrep Kits KAPA Stranded RNA-Seq Kits with RiboErase KAPA RNA HyperPrep Kits with RiboErase
Input Amount 10 ng – 400 ng into library prep 1 ng – 100 ng into library prep 100 ng – 4 µg into mRNA capture 50 ng – 1 µg into mRNA capture 100 ng – 1 µg into rRNA depletion 25 ng – 1 µg into rRNA depletion
Sample Type High-quality total RNA
Degraded or FFPE total RNA
Enriched RNA
High-quality total RNA High-quality total RNA
Degraded or FFPE total RNA
Species Eukaryotic (animal, plant, etc.)
Prokaryotic (bacterial, etc.)
Eukaryotic (animal, plant, etc.) Human, mouse, and rat
Differentiating Applications Targeted RNA-Seq
Whole transcriptome
mRNA-Seq Non-coding RNA
Whole transcriptome
Shared Applications Gene expression analysis; detection of gene fusions, isoforms, and other structural variants;
novel transcript identification; SNV discovery
Strand-specific Yes
Automation friendly Yes

 

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