KAPA mRNA HyperPrep Kits

• Gene expression
• Single nucleotide variation (SNV) discovery
• Splice junction and gene fusion identification
• Characterization of polyadenylated RNAs
   

What is the difference between the KAPA mRNA Hyper Prep Kit and the KAPA Stranded mRNA-Seq Kit?

The KAPA mRNA Hyper Prep Kit offers a streamlined solution to mRNA-Seq library preparation. While much of the workflow is similar to the KAPA Stranded mRNA-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 mRNA Hyper Prep Kit is also provided with KAPA Pure Beads for reaction cleanups.

Yes, the KAPA mRNA Hyper Prep Kit includes mRNA capture reagents for poly(A) mRNA capture.

No, these kits are not compatible with small RNA.

No, KAPA mRNA Hyper Prep Kits are only suitable for mRNA capture and library construction from high-quality input material. The use of fragmented RNA will result in strong bias towards the 3’-end of the mRNA. To determine the quality of RNA, the sample may be analysed using an Agilent Bioanalyzer RNA Kit. RNA with a RIN score less than 7 is not recommended for this protocol.

• mRNA capture using magnetic oligo-dT beads;
• 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.

50 ng – 1 µg of purified total RNA in ≤50 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 mRNA capture through library amplification can be performed in approximately 5.5 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 mRNA capture (steps 2.1–2.19), the resuspended beads (in 22 μL of Fragment, Prime and Elute Buffer) may be stored at 4°C for ≤24 hours.
• 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. The table below outlines various fragmentation parameters depending on the input RNA and the desired insert size.

KAPA Dual-Indexed and Single-Indexed Adapters are recommended for use with KAPA RNA HyperPrep Kits. 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 SeqCap EZ, Illumina TruSeq, Agilent SureSelectXT2, 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 kapabiosystems.com/support.

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 amplification.^1,2,3,4

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 a final amplified library with a concentration of 10 nM to minimize amplification bias. 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.

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. These kits employ primers based on the Illumina flow cell oligos, and can be used to quantify libraries that are ready for flow-cell amplification.

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 mRNA capture reagents and 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. 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. These constructs 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.