KAPA Stranded mRNA-Seq Kits

Overview

The KAPA Stranded mRNA-Seq Kit makes use of KAPA mRNA Capture Beads prior to library preparation to enrich for mRNA over non-polyadenylated species such as ribosomal, precursor or noncoding RNAs.

The kits contain KAPA HiFi DNA Polymerase, selected through our directed evolution technology for high-efficiency and low-bias library amplification and are optimized for the improved coverage of GC-rich and low-abundance transcripts.*  Roche Sequencing Solutions  provides a complete library preparation solution with KAPA Adapters and KAPA Pure Beads, available separately.

Benefits of KAPA Stranded mRNA-Seq Kits

• Focused sequencing of protein-coding transcripts

• Higher coverage of GC-rich transcripts compared to other options*

• Identification of more transcripts and genes compared to other options*

• Qualified automation methods

 

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Product Highlights

Achieve high sequence data quality

  • Improved sequencing throughput with lower duplication rates and increased identification of unique transcripts and genes while maintaining strand specificity compared to other vendor kits
  • Consistent coverage uniformity across transcripts 

Uncover challenging transcripts 

  • Improved coverage of GC-rich transcripts
  • Enhanced identification of exonic regions

Detect low-abundance transcripts

  • Identification of transcripts missed by existing methods, even with lower mRNA input*
  • High uniformity across varying amounts of sample input*

*Data on file.

Kits can be stored for up to 8 months at -20˚C. KAPA mRNA Capture Beads can be stored for up to 8 months at -4˚C.

Specifications
Spec
Description
Compatible Platform
Illumina HiSeq,NextSeq,MiSeq and GAIIX
Starting Material
Total RNA,mRNA, or ribosomal depleted RNA
Input Amount 100 ng - 4 µg 

Components

Components

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Kits include reagents for RNA fragmentation, 1st strand cDNA synthesis and 2nd strand synthesis/marking, and cDNA library preparation, including A-tailing, ligation and library amplification. Kits with reagents for mRNA capture are also available.

Kit Code Roche Cat No. Description Kit Size How to Buy
KK8420
07962193001
KAPA Stranded mRNA-Seq Kit, with KAPA mRNA Capture Beads
24 libraries Contact Us
KK8421
07962207001
KAPA Stranded mRNA-Seq Kit, with KAPA mRNA Capture Beads
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
KK8000
07983271001
KAPA Pure Beads (5 mL)
5 mL
Contact Us
KK8001
07983280001
KAPA Pure Beads (30 mL)
30 mL
Contact Us
KK8002
07983298001
KAPA Pure Beads (60 mL) 60 mL
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
  • Gene expression
  • Polymorphism detection
  • Genome annotation
  • Alternative splicing
  • RNA editing

No, these kits are not compatible with small RNA.

100 ng–4 µg of purified total RNA, dissolved in <50 µL RNase-free water.

  • mRNA capture using magnetic oligo-dT beads
  • Fragmentation using heat and magnesium
  • 1st Strand cDNA Synthesis using random priming
  • 2nd Strand cDNA Synthesis and marking, which converts the cDNA:RNA hybrid to double-stranded cDNA (dscDNA) and incorporates dUTP in the second cDNA strand
  • A-tailing to add 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
  • 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.

Yes, the KAPA Stranded mRNA-Seq Kits include the KAPA mRNA capture beads required for poly(A) mRNA capture.

Yes, during 2nd strand synthesis, the DNA: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 RNA fragmentation through library amplification can be performed in 6-8 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, resuspend the beads in 22 µL of Fragment, Prime, and Elute Buffer, and store the sealed tube at 4°C for up to 24 hours.
  • After the 2nd strand synthesis cleanup, resuspend the washed beads in 15 µL of 1x A-Tailing Buffer (without enzyme) and store the sealed tube at 4°C for up to 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 up to 24 hours, or at -20°C for up to 1 week.

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 Adapters are recommended for use with KAPA Stranded mRNA-Seq Kits. However, kits are also compatible with non-indexed, single-indexed, and dual-indexed adapters that are routinely used in Illumina TruSeq, SeqCap EZ, Agilent SureSelect, and other similar library construction and target capture 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.

 

While it is not necessary to adjust adapter concentrations to accommodate moderate sample-to-sample variation, an adapter concentration appropriate for the amount of input RNA is recommended. The table below summarizes recommended adapter concentrations for various inputs into the mRNA capture reaction.

 

Quantity of starting material Adapter stock concentration Adapter concentration in ligation reaction
2001 – 4000 ng 1400 nM 100 nM
501 – 2000 ng 700 nM 50 nM
251 – 500 ng 350 nM 25 nM
100 – 250 ng 140 nM 10 nM

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.

KAPA 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 formatin 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 pooled and sequenced on a 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.

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.

 

Desired Insert Size Temperature Duration
100-200 bp 94˚C 8 min
200-300 bp 94˚C 6 min
300-400 bp 85˚C 6 min

The size distribution of the double-stranded cDNA and/or final amplified library should be confirmed with an electrophoretic method. The quantification of the library should be done with a qPCR based quantification kit such as the KAPA Library Quantification Kits 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.

KAPA HiFi HotStart 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

  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).

To minimize over-amplification and associated unwanted artifacts, the number of PCR cycles should be optimized to produce a final amplified library with a concentration range of 10-30 ng/µL which is equivalent to 0.5-1.5 µg of DNA per 50 µL reaction. The number of cycles recommended below should be used as a guide for library amplification, but cycle numbers may have to be adjusted (± 4 cycles) depending on library amplification efficiency, RNA fragmentation profile, and the presence of adapter dimers.

 

Input RNA Number of Cycles
100–250 ng 10–16
251–500 ng 10–14
501–2000 ng 8–12
2001–4000 ng 6–10

KAPA Stranded mRNA-Seq Kits are supplied in two boxes. Box 1 contains capture beads and buffers, and is shipped at 4˚C. The contents of Box 1 must not be frozen, as this will damage beads. Upon receipt, store Box 1 at 4˚C. Box 2 contains enzymes and buffers for cDNA synthesis and library preparation, and is shipped on dry ice or ice packs, depending on the destination country. The contents of Box 2 are temperature-sensitive, and appropriate care should be taken during storage. Upon receipt, store Box 2 at -20˚C in a constant temperature freezer. The 1st Strand Synthesis Buffer and PEG/NaCl Solution supplied in Box 2 are light-sensitive, and should be protected from light during storage. When stored under these conditions and handled correctly, the kit components will retain full activity until the expiry date indicated on the kit label.