KAPA RNA HyperPrep Kits

Overview

The KAPA RNA HyperPrep Kits utilize a novel chemistry that enables the combination of enzymatic steps and fewer reaction purifications, resulting in a truly streamlined solution for the preparation of high-quality RNA-seq libraries. The strand-specific workflow is flexible, supporting library construction from lower-input amounts and degraded samples and is compatible with mRNA capture and ribosomal RNA and/or globin transcript depletion. 

Every KAPA RNA HyperPrep Kit includes the same core library construction chemistry with an optional upfront depletion module, making up four workflow solutions: either mRNA capture, rRNA depletion, rRNA and globin depletion or none. Roche Sequencing Solutions provides a complete library preparation solution with KAPA Pure Beads (included) and KAPA Dual-Indexed Adapters (available separately). These kits are designed to be automation-friendly on a majority of commercially available liquid handlers.

 

 

Features and Benefits of KAPA RNA HyperPrep Kits
  • Reduced hands-on and overall time through fewer enzymatic and reaction cleanup steps* 
  • Strand-specific, sequencing-ready libraries from input RNA in ~4 hours*
  • Single-day library construction, including mRNA capture and rRNA depletion with the KAPA RNA HyperPrep workflow*
  • Robust performance across different sample types and input amounts*
  • High throughput and consistency with an automation-friendly workflow
  • Validated KAPA RNA HyperPrep Kit data analysis solutions provided through the Genialis platform 
KAPA RNA HyperPrep Kit.jpg
Learn more about the Roche and Genialis collaboration
Features and Benefits of KAPA RNA HyperPrep Kits
  • Reduced hands-on and overall time through fewer enzymatic and reaction cleanup steps* 
  • Strand-specific, sequencing-ready libraries from input RNA in ~4 hours*
  • Single-day library construction, including mRNA capture and rRNA depletion with the KAPA RNA HyperPrep workflow*
  • Robust performance across different sample types and input amounts*
  • High throughput and consistency with an automation-friendly workflow
  • Validated KAPA RNA HyperPrep Kit data analysis solutions provided through the Genialis platform 
KAPA RNA HyperPrep Kit.jpg
Product Highlights

Flexible workflow options for a variety of applications

  • Stranded library construction solutions for sequencing of both coding and non-coding transcripts
  • Compatible with a variety of sample types and input amounts, of both high-quality and degraded RNA
Learn about working with degraded inputs and tumor profiling applications. Presented by Dr. Nancy Nabilsi
Single-day, single-tube library prep
  • Reduce hands-on and overall turnaround time with fewer enzymatic and cleanup steps
  • Produce strand-specific libraries from input RNA in approximately 4 hours
  • Complete the entire workflow, inclusive of upfront RNA enrichment, in a standard work day
  • Achieve high throughput and consistency with automation-friendly workflows

Sequence what matters

  • Effective RNA enrichment (rRNA depletion or mRNA capture), combined with highly efficient library construction, reduces the number of reads associated with unwanted content and PCR duplicates
  • More unique transcripts and genes are detected from a fixed amount of sequencing

Cover your bases with higher uniformity

  • Efficient RNA enrichment and library construction processes results in more even coverage along transcript lengths
  • Library amplification with KAPA HiFi enables better coverage of difficult GC-rich region

Streamlined end-to-end RNA library preparation and analysis workflow

  • By partnering with Genialis to develop an automated, data analysis and visualization solution validated for KAPA RNA HyperPrep Kits, Roche Sequencing Solutions provides customers with an end-to-end RNA workflow that is simple, proven and complete.
  • Allows every processing step to be traced to ensure quality and reproducibility and management of data, projects and collaborations in the cloud. 

 

*Data on file.

For Research Use Only. Not for use in diagnostic procedures.
KAPA is a trademark of Roche. Other product names and trademarks are the property of their respective owners.

The table below describes the specifications for the KAPA RNA HyperPrep Kits, including all four workflows:

For Research Use Only. Not for use in diagnostic procedures.
KAPA is a trademark of Roche. Other product names and trademarks are the property of their respective owners.

For Research Use Only. Not for use in diagnostic procedures.
KAPA is a trademark of Roche. Other product names and trademarks are the property of their respective owners.

KAPA RNA HyperPrep Kits include all reagents required for RNA fragmentation, cDNA synthesis, library preparation and amplification. KAPA Pure Beads are supplied in each kit for reaction cleanups, while KAPA Adapters are available separately. Kits also include upfront depletion modules for mRNA capture (KK8580, KK8581), rRNA depletion (KK8560, KK8561), and rRNA and globin depletion (KK8562, KK8563).

Kit Code
Roche Cat. No
Description
Kit Size
How to buy
KK8540
08098093702
KAPA RNA HyperPrep Kit
24 libraries
Contact Us
KK8541
08098107702
KAPA RNA HyperPrep Kit
96 libraries
Contact Us
KK8580 08098115702
KAPA mRNA HyperPrep Kit 24 libraries Contact Us
KK8581 08098123702
KAPA mRNA HyperPrep Kit
96 libraries
Contact Us
KK8560 08098131702
KAPA RNA HyperPrep Kit with RiboErase (HMR)
24 libraries
Contact Us
KK8561 08098140702
KAPA RNA HyperPrep Kit with RiboErase (HMR)
96 libraries
Contact Us
KK8562 08308314702 KAPA RNA HyperPrep Kit with RiboErase (HMR) Globin
24 libraries
Contact Us
KK8563 08308241702 KAPA RNA HyperPrep Kit with RiboErase (HMR) Globin
96 libraries
Contact Us

Accessory Products


KAPA Adapter Kits contain KAPA Adapter Dilution Buffer (25 mL) as well as three additional sealing films to support multiple use. KAPA Adapter Dilution Buffer (08278539001) is also available separately, if required.

Kit Code
Roche Cat. No
Description
Kit Size
How to buy
KK8727 08861919702 KAPA Unique Dual-Indexed Adapters Kit (15 μM) 96 adapters x 20 μL each Contact Us
KK8722
08278555702
KAPA Dual-Indexed Adapter Kit, (15 µM)
96 adapters x 20 µl each
Contact Us
KK8721
08278539001
KAPA Adapter Dilution Buffer (25 mL)
25 mL Contact Us

For Research Use Only. Not for use in diagnostic procedures.
KAPA is a trademark of Roche. Other product names and trademarks are the property of their respective owners.

1. General

The poly(A) capture method used in an mRNA capture workflow is useful when specifically interrogating mRNA species, however the workflow does bias towards exonic transcripts. The poly(A) capture approach also tends to have reduced 5' coverage of transcripts, due to the 3' polyadenylation of mRNA transcripts.

The use of the KAPA RNA HyperPrep Kits with RiboErase (HMR) and/or RiboErase (HMR) Globin will allow a more accurate representation of the whole transcriptome and will contain all RNA species except rRNA and/or globin. This workflow retains intronic and intergenic regions, which is where many long non-coding transcripts are found. Additionally, the poly(A) capture approach makes it incompatible for use with degraded RNA, where there is the possibility of strand breaks between the 3' polyadenylated region and the rest of the transcript.

 

If you are using one of our KAPA Stranded RNA-Seq Kits, either with mRNA capture or rRNA and/or globin transcript depletion, please visit our online resource for Technical Documentation or alternatively, please visit sequencing.roche.com/contactus.

2. Compatibility

No, these kits are not compatible with small RNA.

The KAPA RNA HyperPrep Kits (with no upfront enrichment) and the KAPA RNA HyperPrep Kits with RiboErase (HMR) and/or RiboErase (HMR) Globin are compatible with RNA extracted from formalin-fixed paraffin embedded (FFPE) tissue. The quality of FFPE-derived RNA can be highly variable due to the damaging nature of the formalin fixation process, where crosslinking, chemical modification, and fragmentation can occur. Library construction results may vary depending on the input amount and quality of the RNA. Higher RNA inputs may salvage library construction for particularly difficult FFPE samples. Please refer to the recommendations outlined in the Technical Data Sheet for each kit.

KAPA mRNA Hyper Prep Kits are only suitable for mRNA capture and library construction from high-quality input material (RIN ≥ 7). The use of fragmented RNA will result in strong bias towards the 3’-end of the mRNA.

 

 

The KAPA RNA HyperPrep Kits (with no upfront enrichment) are compatible with any species. The KAPA mRNA HyperPrep Kits are compatible with any species with
3’ poly-A tails.

The KAPA RNA HyperPrep Kits with RiboErase (HMR) or RiboErase (HMR) Globin are compatible with human, mouse, and rat rRNA and/or globin transcript depletion.


 

Yes, both cytoplasmic and mitochondrial rRNA is depleted.

Both the KAPA RNA HyperPrep Kit with RiboErase (HMR) and the KAPA RNA HyperPrep Kit with RiboErase (HMR) Globin use the same workflow and reagents, which allows for depletion of rRNA from human, mouse and rat species. The only difference is that an additional tube of depletion oligos are supplied and added during the initial hybridization step of the workflow, to deplete globin transcripts when using the KAPA RNA HyperPrep Kit with RiboErase (HMR) Globin. These globin depletion oligos target globin mRNA derived from hemoglobin alpha 1 (HGA1), hemoglobin alpha 2 (HGA2), hemoglobin beta (HGB), and hemoglobin gamma (HGG).
 

3. Workflow

  • Fragmentation using heat and magnesium;
  • 1st strand cDNA synthesis using random priming;
  • Combined 2nd Strand cDNA Synthesis and A-tailing, which converts the cDNA:RNA hybrid to double-stranded cDNA (dscDNA), incorporates dUTP in the second cDNA strand and adds dAMP to the 3′-ends of the dscDNA library fragments;
  • Adapter ligation, where dsDNA adapters with 3′-dTMP overhangs are ligated to A-tailed library insert fragments; and
  • Library amplification to amplify library fragments carrying appropriate adapter sequences at both ends using high-fidelity, low-bias PCR. The strand marked with dUTP is not amplified, allowing strand specific sequencing.
     

The upfront enrichment module for mRNA capture make use of magnetic oligo-dT beads. The rRNA and/or globin depletion include hybridization of complementary DNA oligonucleotides, followed by treatment with RNase H and DNase to remove rRNA and/or globin transcripts duplexed to DNA and original DNA oligonucleotides, respectively.
 

KAPA Pure Beads are provided in this kit for reaction purification steps. It is a suspension of paramagnetic beads in a buffer optimized for purification in next-generation sequencing and other molecular biology workflows. KAPA Pure Beads are compatible with manual processing or automated liquid handling and enables efficient recovery in both formats.

Yes, during 2nd strand synthesis, the cDNA:RNA hybrid is converted to dscDNA, with dUTP incorporated into the second cDNA strand. During library amplification, the strand containing dUTP is not amplified, allowing strand-specific sequencing. This kit retains accurate strand origin information in ˃99% of unique mapped reads.

The sample preparation process may be paused safely after the upfront depletion modules as follows:
After mRNA capture, the resuspended beads (in 22 μL of Fragment, Prime and Elute Buffer) may be stored at 4°C for ≤24 hours.
After rRNA and/or globin depletion, following elution in 1X Fragment, Prime and Elute Buffer, samples may be stored at -20°C for ≤24 hours.

The library construction process from RNA fragmentation through library amplification can be performed in approximately 4 hours, depending on the number of samples being processed, and experience. If necessary, the protocol may be paused safely after any of the following steps:

  • After the first post-ligation cleanup, store the resuspended beads at 4°C for up to 24 hours. Do not freeze the beads, as this can result in dramatic loss of DNA.
  • After the second post-ligation cleanup, store the eluted, unamplified library DNA at 4°C for ≤1 week, or at -20°C for ≤1 month.

 

RNA is fragmented using high temperature in the presence of magnesium. Depending on the origin and integrity of the input RNA, and the intended application, different RNA fragmentation protocols are provided to obtain the required insert size distribution. For intact RNA, such as that extracted from fresh/frozen tissue, longer fragmentation is required at higher temperatures. For degraded or fragmented RNA (e.g. from older samples or FFPE tissue), use a lower temperature and/or shorter times. The Technical Data Sheet for each product outlines various fragmentation parameters depending on the input RNA and the desired insert size.

KAPA Dual-Indexed Adapters are recommended for use with the KAPA RNA Hyper Prep Kits. However, these workflows are also compatible with other full-length adapter designs wherein both the sequencing and cluster generation sequences are added during the ligation step, such as those routinely used in Illumina TruSeq, SeqCap EZ, Agilent SureSelect XT2, and other similar library construction workflows.

Custom adapters that are of similar design and are compatible with “TA-ligation” of dsDNA may also be used, remembering that custom adapter designs may impact library construction efficiency. Truncated adapter designs, where cluster generation sequences are added during amplification instead of ligation, may require modified post-ligation cleanup conditions. For assistance with adapter compatibility, please visit sequencing.roche.com/contactus.

Please refer to the KAPA Dual-Indexed Adapters Technical Data Sheet for information about barcode sequences, pooling, kit configurations, formulation, and dilution for the different KAPA RNA HyperPrep Kits and inputs.

Purified, adapter-ligated cDNA can be stored at 4°C for one week or at -20°C for at least one month, before amplification and/or sequencing. To avoid degradation, always store DNA in a buffered solution (10 mM Tris-HCl, pH 8.0) and minimize the number of freeze-thaw cycles.

KAPA HiFi HotStart DNA Polymerase is the enzyme in the KAPA HiFi HotStart ReadyMix, provided in the KAPA RNA HyperPrep Kits. This is a novel B-family DNA polymerase engineered for low-bias, high fidelity PCR and is the reagent of choice for NGS library amplification1,2,3,4.

  1. Oyola, S.O. et al. BMC Genomics 13, 1 (2012).
  2. Quail, M.A. et al. Nature Methods 9, 10–11 (2012).
  3. Quail, M.A. et al. BMC Genomics 13, 341 (2012).
  4. Ross, M.G. et al. Genome Biology 14, R51 (2013).

To minimize over-amplification and associated unwanted artefacts, the number of PCR cycles should be optimized to produce enough final, amplified library for downstream analysis and quality control, based on the Illumina instrument requirements. For target capture workflows, typically 1 µg of library yield is required, which may differ depending on the method used and the pre-capture multiplexing strategy employed.

The number of cycles recommended in the Technical Data Sheets for each KAPA RNA HyperPrep Kit should be used as a guide for library amplification, but cycle numbers may have to be adjusted depending on desired final library yield, library amplification efficiency, RNA fragmentation profile, and the presence of adapter dimers.

The size distribution of the dscDNA and/or final amplified library should be confirmed with an electrophoretic method. The quantification of the library should be performed with a qPCR based quantification kit such as the KAPA Library Quantification Kit for Illumina platforms. This kit employs primers based on the Illumina flow cell oligos, and can be used to quantify libraries that are ready for flow-cell amplification.

4. Data Analysis

Roche Sequencing Solutions has partnered with Genialis to provide a data analysis and visualization platform, validated specifically for KAPA RNA HyperPrep Kits. This provides our customers with an end-to-end RNA workflow that is simple, proven and complete. This cloud-based data analysis solution, allows every processing step to be traced to ensure quality and reproducibility and management of data, projects and collaborations.

Benefits of the Genialis platform include:

  • Establishing reproducibility across projects by running validated, single-click bioinformatics pipelines through cloud-hosted computational resources so that your primary data analyses are performed the same way across projects.
  • Verifying consistency between your experimental replicates and identifying relevant correlations or differences amongst experimental conditions, to ensure design quality.
  • Comparison of expression levels across conditions by quantifying gene abundance in individual samples and investigate differential expression of individual genes, pre-defined gene sets, and entire transcriptomes.
  • Understand how specific experimental conditions affect the regulation of groups of related genes and their corresponding biological functions.

5. Storage and Quality Control Information

The KAPA RNA HyperPrep Kits are supplied in multiple boxes.

  • The components for cDNA synthesis and library preparation are temperature sensitive, and should be stored at -15°C to -25°C in a constant-temperature freezer upon receipt. The PEG/NaCl Solution may be stored at 4°C for up to 2 months or at -20°C until expiry date. 
  • The upfront enrichment module for mRNA capture, should be stored at 2°C to 8°C and for rRNA and/or globin depletion should be stored at -15°C to -25°C upon receipt. 
  • Store KAPA Pure Beads at 2°C to 8°C.

When stored under these conditions and handled correctly, the kit components will retain full activity until the expiry date indicated on the kit label.
 

KAPA Dual-Indexed Adapters undergo extensive qPCR- and sequencing-based functional and QC testing to confirm:

  • optimal library construction efficiency
  • minimal levels of adapter-dimer formation
  • nominal levels of barcode cross-contamination

Library construction efficiency and adapter-dimer formation are assessed in a low-input library construction workflow. Library construction efficiency is calculated by measuring the yield of adapter-ligated library (before any amplification) by qPCR (using the KAPA Library Quantification Kit), and expressing this as a % of input DNA. To assess adapter-dimer formation, a modified library construction protocol— designed to measure adapter dimer with high sensitivity—is used. Pass criteria for this assay translate to adapter-dimer carry-over in a standard workflow in the range of 0 – 2%.

Barcode cross-contamination is assessed by sequencing. Each adapter is ligated to a unique, synthetic insert of known sequence using a standard library construction protocol. Libraries are pooled and sequenced on a MiSeq™. For every barcode, the number of reads (at least 115,000) associated with each of the 96 inserts is counted, and the total % of correct inserts calculated. Contamination of any barcode with any other single barcode is guaranteed to be <0.25%. The total level of contamination for any barcode is typically in the range of 0.1 – 0.5%. This assay is unable to distinguish between chemical cross-contamination and adapter “cross-talk”, and measures the total number of incorrect inserts resulting from both phenomena. Adapter "cross-talk" occurs when a barcode is "misread" during sequencing and subsequent analysis. The propensity for "cross-talk" differs for different adapter pairs and depends on how closely the barcode sequences are related. 

KAPA Adapter Dilution Buffer is free of detectable contaminating exo- and endonuclease activities, and meet strict requirements with respect to DNA contamination.

 

For Research Use Only. Not for use in diagnostic procedures.
KAPA is a trademark of Roche. Other product names and trademarks are the property of their respective owners.