KAPA RNA HyperPrep Kits with RiboErase (HMR)

Single-Day RNA

The KAPA RNA HyperPrep Kits with RiboErase (HMR) utilize 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 rRNA-depleted RNA-seq libraries. By utilizing a targeted enzymatic method for depletion, our workflow enables efficient reduction of ribosomal RNA (rRNA) and a more complete representation of the transcriptome, including precursor mRNAs and non-coding RNA (ncRNA). The strand-specific workflow is flexible – supporting library construction from lower-input amounts and degraded samples. Kits contain all reagents required for rRNA depletion and library preparation, with the exception of KAPA Adapters (available separately). Benefits include:

  • single-day library construction, inclusive of rRNA depletion
  • reduced hands-on and overall time through fewer enzymatic and reaction cleanups
  • flexible input of 25 ng – 1 µg total RNA from human, mouse, or rat species*
  • higher success rates with lower input and degraded samples
  • maintain over 99% strand specificity*
  • KAPA Pure Beads included for reaction purifications

NEW! KAPA Single-Indexed Adapter Kits are now available. For more information on the appropriate Adapter Kit for your library construction workflow and input, scroll down to the Ordering section, or download the KAPA Single-Indexed Adapter Calculator. Download our KAPA Single-Indexed Adapter Kit Calculator *Data on file. For Research Use Only. Not for use in diagnostic procedures.

Product Highlights

Sequence what matters

  • Waste fewer reads due to the combination of rRNA carryover and PCR duplicates
  • Identify more unique transcripts and genes with equivalent sequencing

Achieve superior coverage uniformity

  • Obtain more uniform distribution of reads across transcripts
  • Improve coverage of difficult GC-rich transcripts

Generate high-quality libraries from degraded samples

  • Input as little as 25 ng with FFPE samples, depending on total RNA quality
  • Achieve low duplication rates and highly efficient, reproducible rRNA removal with degraded samples
  • Identify more unique transcripts and genes with equivalent sequencing

 

Achieve reliable results with degraded inputs

  • Attain a high-degree of expression correlation between paired FFPE and fresh frozen samples which provides increased confidence in sequence data accuracy

Related Products

Are you sequencing low-input, FFPE or high quality DNA?  RNA?  Check out these Kapa NGS products to improve your workflow and results:

KAPA mRNA HyperPrep Kits

KAPA mRNA HyperPrep Kits

KAPA Stranded mRNA-Seq Kits

KAPA Stranded mRNA-Seq Kits

Library Amplification

KAPA Library Amplification Kits

Library Preparation

KAPA HyperPrep Kits

Library Quantification

KAPA Library Quantification Kits

Applications
  • Whole transcriptome
  • Gene expression analysis
  • Detection of gene fusions, isoforms, and other structural variants
  • Novel transcript identification, including noncoding transcripts
  • SNV discovery
Kit Specifications and Contents / Storage

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

Kits contain Hybridization Buffer, Hybridization Oligos (HMR), Depletion Buffer, RNase H, DNase Buffer, DNase, Fragment, Prime and Elute Buffer (2X), 1stStrand Synthesis Buffer, KAPA Script, 2nd Marking Buffer, 2nd Strand Synthesis & A-Tailing Enzyme Mix, Ligation Buffer, DNA Ligase, PEG/NaCl Solution, KAPA Pure Beads, Library Amplification Primer Mix (10X), and KAPA HiFi HotStart ReadyMix (2X).

RNAHyperRibo_Component Chart

Specifications

Spec
Description
Compatible Platform
0616
Library Type
RNA
Starting Material
High-quality and degraded total RNA
Input Amount
25 ng – 1 µg
FAQs

1. General

What is the difference between the KAPA RNA HyperPrep Kit with RiboErase (HMR) and the Stranded RNA-Seq Kit with RiboErase (HMR)?

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.

Are poly(A) capture beads included in the kits?

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.

Why would I use this kit over an mRNA-Seq method?

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.

 

2. Compatibility

Are these kits compatible with small RNA library preparation?

No, these kits are not compatible with small RNA.

Are these kits compatible with FFPE-derived 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.

With which species is this kit compatible?

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.

While the DNA oligos used for depletion were specifically designed to target human, mouse, and rat rRNA sequences, there is potential for the kit to be used with other species where high homology to human, mouse, or rat rRNA sequences is observed. Please contact kapabiosystems.com/support with specific species inquiries.

 

3. Workflow

What are the major steps in library construction?

  • 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

What are the input requirements of the KAPA RNA HyperPrep Kits with RiboErase (HMR)?

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

Does this kit deplete globin?

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

What are KAPA Pure Beads?

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.

Do the kits offer strand-specific information?

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.

Are there safe stopping points in the sample preparation process?

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.

What method of RNA fragmentation does this kit make use of?

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.

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

What adapters can I use with this kit?

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, Roche 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.

Where do I find more information about KAPA Single-Indexed Adapters?

Please refer to the KAPA Single-Indexed Adapter Technical Data Sheet (include link) for information about barcode sequences, pooling, kit configurations, formulation, and dilution of KAPA Single-Indexed Adapters.

How long can adapter-ligated cDNA be stored?

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.

Which polymerase is used for amplification?

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, 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).

How many cycles should I use when amplifying my adapter-ligated library?

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.

Input RNA Number of Cycles
25 – 100 ng 11 – 15
101 – 250 ng 9 – 12
251 – 500 ng 7 – 10
501 – 1000 ng 6 – 8

How should I measure the final library?

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.

 

4. Storage and Quality Control Information

What are the storage conditions for this kit?

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.

What QC testing is performed on KAPA Single-Indexed Adapters?

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.

Ordering
Kit Code
Roche Cat. No
Description
Kit Size
How to buy
KK8560
08098131702
Complete Kit
24 libraries
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KK8561
08098140702
Complete Kit
96 libraries
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Accessories

Adapter Set A contains indices 2, 4, 5, 6, 7, 12, 13, 14, 15, 16, 18 and 19. Adapter Set B contains indices 1, 3, 8, 9, 10, 11, 20, 21, 22, 23, 25, 27. All kits contain KAPA Adapter Dilution Buffer.

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
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KK8702
08005729001
KAPA Single-Indexed Adapter Kit, Set B (30 µM)
12 adapters x 40 µl each
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KK8710
08005770001
KAPA Single-Indexed Adapter Kit, Set A + B (1.5 µM)
24 adapters x 40 µl each
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KK8711
08005788001
KAPA Single-Indexed Adapter Kit, Set A (1.5 µM)
12 adapters x 40 µl each
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KK8712
08005796001
KAPA Single-Indexed Adapter Kit, Set B (1.5 µM)
12 adapters x 40 µl each
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for pricing