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 depletion. KAPA RNA HyperPrep Kits contain all reagents required for RNA library preparation, with the exception of KAPA Adapters (available separately).

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

*Data on file.

Enzymes and buffers for rRNA depletion, cDNA synthesis, and library preparation can be stored for up to 10 months at -20°C. mRNA capture reagents and KAPA Pure Beads can be stored for up to 10 months at 4°C. (US only)

Kits include reagents for RNA fragmentation, cDNA synthesis, and library preparation. Kits with reagents for mRNA capture are also available.

Specifications
Spec
Description
Compatibility Platform
Illumina HiSeq, NextSeq, and MiSeq
Library Type
RNA
Starting Material High-quality total RNA, mRNA, or rRNA-depleted RNA
Input Amount  1 ng – 100 ng

Components

Kits include reagents for RNA fragmentation, cDNA synthesis, and library preparation. Kits with reagents for mRNA capture are also available.

Kit Code
Roche Cat. No
Description
Kit Size
How to buy
KK8540
08098093702
KAPA RNA HyperPrep Kit
24 libraries
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KK8541
08098107702
KAPA RNA HyperPrep Kit
96 libraries
Contact Us

Accessory Products

KAPA Single-Indexed Adapter Set A contains indices 2, 4, 5, 6, 7, 12, 13, 14, 15, 16, 18 and 19, whereas Set B contains indices 1, 3, 8, 9, 10, 11, 20, 21, 22, 23, 25, 27. All KAPA Single- and Dual-Indexed Adapter Kits contain KAPA Adapter Dilution Buffer. KAPA Dual-Indexed Adapter Kits also contain three additional sealing films to support multiple use.

Kit Code
Roche Cat. No
Description
Kit Size
How to buy
KK8700
08005699001
KAPA Single-Indexed Adapter Kit, Set A + B (30 µM)
24 adapters x 40 µl each
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KK8701
08005702001
KAPA Single-Indexed Adapter Kit, Set A (30 µM)
12 adapters x 40 µl each
Contact Us
KK8702
08005729001
KAPA Single-Indexed Adapter Kit, Set B (30 µM)
12 adapters x 40 µl each
Contact Us
KK8710
08005770001
KAPA Single-Indexed Adapter Kit, Set A + B (1.5 µM)
24 adapters x 40 µl each Contact Us
KK8711
08005788001
KAPA Single-Indexed Adapter Kit, Set A (1.5 µM)
12 adapters x 40 µl each
Contact Us
KK8712
08005796001
KAPA Single-Indexed Adapter Kit, Set B (1.5 µM)
12 adapters x 40 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

1. General

  • Gene expression
  • Polymorphism detection
  • Genome annotation
  • Alternative splicing

KAPA RNA HyperPrep Kits offers a streamlined solution to RNA-Seq library preparation. While much of the workflow is similar to the KAPA Stranded RNA-Seq Library Preparation Kit, the main differences are a combined 2nd strand synthesis and A-tailing reaction, which reduces the total number of enzymatic steps and shortens the workflow by 1 hour and 20 minutes. In addition, a bead purification step has been removed, further reducing hands-on time and overall workflow time by 20 minutes. The KAPA RNA HyperPrep Kit is also provided with KAPA Pure Beads for reaction cleanups.

 

No, KAPA RNA HyperPrep Kits do not include beads for mRNA capture. For applications which require mRNA enrichment, we would recommend the KAPA mRNA HyperPrep Kit, as it includes KAPA mRNA capture reagents.

2. Compatibility

No, these kits are not compatible with small RNA.

The quality of RNA extracted from formalin-fixed paraffin embedded (FFPE) tissue 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 (with a maximum of 100 ng) may salvage library construction for particularly difficult FFPE samples. Please refer to the recommendations outlined in the Technical Data Sheet.

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.

1 – 100 ng of purified RNA (e.g. mRNA captured, rRNA-depleted, or total RNA) in ≤10 µL of RNase-free water.

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 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 table below outlines various fragmentation parameters depending on the input RNA and the desired insert size.

 

Input RNA Desired Insert Size Fragmentation and Priming
Intact 100 – 200 bp 8 min @ 94˚C
200 – 300 bp 6 min @ 94˚C
300 – 400 bp 6 min @ 85˚C
Partially degraded 100 – 300 bp 16 min @ 85˚C
Degraded * 100 – 200 bp 1 min @ 65˚C

 

* This facilitates annealing of the random primers, and will not result in any significant additional fragmentation of the RNA.

KAPA Single-Indexed Adapters are recommended for use with the KAPA RNA Hyper Prep Kit. However, this workflow is 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.html.

Please refer to the KAPA Single-Indexed and Dual-Indexed Adapter Technical Data Sheets for information about barcode sequences, pooling, kit configurations, formulation, and dilution for different KAPA DNA and RNA library preparation 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 provided in the KAPA HiFi HotStart ReadyMix. 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, 1011 (2012).

3. Quail, M.A. et al. BMC Genomics 13, 341 (2012).

4. Ross, M.G. et alGenome Biology 14, R51 (2013).

To minimize over-amplification and associated unwanted artefacts, the number of PCR cycles should be optimized to produce a final amplified library with a concentration of 10 nM to minimize amplification bias. For 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 below 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.

 

Input RNA Number of Cycles
1 – 5 ng 11 – 14
6 – 20 ng                   9 – 12
21 – 50 ng 7 – 10
51 – 100 ng 6 – 8

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. Storage and Quality Control Information

This kit is 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. Store KAPA Pure Beads at 2°C to 8°C. The PEG/NaCl Solution may be stored at 4°C for up to 2 months or at -20°C until expiry date. 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 Single-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. The conversion rate achieved in the assay indicates library construction efficiency. This 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.

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. These constructs are pooled and sequenced on an Illumina MiSeq. For every barcode, the number of reads (in the range of 115,000–500,000) associated with each insert is counted, and the total % 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..