KAPA RNA HyperPrep Kits with RiboErase

• Gene expression analysis of high- and low-quality RNA samples (e.g., extracted from FFPE tissue)
• Single nucleotide variation (SNV) discovery
• Splice junction and gene fusion identification
• Characterization of both polyadenyated and non-polyadenylated RNAs, including noncoding and immature RNAs

The KAPA RNA HyperPrep Kit with RiboErase (HMR) 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 with RiboErase (HMR), 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 with RiboErase (HMR) is also provided with KAPA Pure Beads for reaction cleanups.

No, the KAPA RNA HyperPrep Kit with RiboErase (HMR) does not include beads for mRNA capture. For applications which require mRNA enrichment, we would recommend the KAPA mRNA HyperPrep Kit (include link), as it includes mRNA capture reagents for mRNA enrichment.

While the poly(A) capture method used in an mRNA-Seq workflow is useful when specifically interrogating mature mRNA species, the workflow does bias towards exonic transcripts. If you wish to obtain a more accurate representation of the whole transcriptome, with only the rRNA sequences removed, then ribodepletion is the better option. This results in retention of data generated from intronic and intergenic regions, which is where many long non-coding transcripts are found.
 

Additionally, the poly(A) capture approach is not recommended for use with degraded RNA, as the possibility of strand breaks between the 3’ polyadenylation and the rest of the transcript is higher.

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 1000 ng) may salvage library construction for particularly difficult FFPE samples. Please refer to the recommendations outlined in the Technical Data Sheet.

The DNA oligos used for depletion were specifically designed with human, mouse, and rat rRNA sequences in mind, and the kit has only been validated for these species.

• Depletion of rRNA by hybridization of complementary DNA oligonucleotides, followed by treatment with RNase H and DNase to remove rRNA duplexed to DNA and original DNA oligonucleotides, respectively;
• 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

25 ng – 1 µg of purified total RNA in ≤10 µL of RNase-free water.

Yes!

Unfortunately, we do not offer a globin depletion solution at this time.

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 double-stranded DNA, 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 rRNA depletion through library amplification can be performed in approximately 6.5 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 elution in 1X Fragment, Prime and Elute Buffer (step 6.5) store the rRNA-depleted material at -20°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. 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 formalin-fixed-paraffin-embedded (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.
* 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 HyperPrep Kit with RiboErase (HMR). 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 kapabiosystems.com/support.

Please refer to the KAPA Single-Indexed  and Dual-Indexed Adapter Technical Data Sheet 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 rRNA depletion, 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. 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.