KAPA Library Preparation Kits for Ion Torrent

It’s complex, but we have it covered.

KAPA DNA Library Preparation Kits for Ion Torrent™ sequencing platforms contain evolved and optimally formulated enzymes that enable the highest overall coverage from the least amount of total sequencing.

Kits are optimized to achieve significantly higher library yields and contain KAPA HiFi for high-fidelity, high-efficiency and low-bias library amplification. This ensures the highest library complexity and sequence data quality, particularly from low input and difficult samples.

 

For Research Use Only. Not for use in diagnostic procedures.

Product Highlights

Data on file.

Achieve greater library complexity

  • Higher yields of adapter-ligated library translates to higher library
    complexity
  • Fewer cycles of amplification are needed, which results in lower duplication rates
  • PCR-free workflows possible from inputs ≥100 ng*
The error rate of KAPA HiFi is calculated at 1 error in 3.54 x 105 bases covered (2.82 x 10-7). The error rate of Kapa HiFi is 100X lower than Taq polymerase, 40X lower than polymerase blends, and 2X lower than Phusion®. Data on file.

Reduced amplification bias and improve coverage

  • KAPA HiFi DNA Polymerase is the gold standard enzyme for high-yield, low-bias library amplification, with a 40X lower error rate than Platinum® Taq HiFi*
  • KAPA HiFi reduces PCR bias and improves coverage uniformity of GC- and AT-rich regions, promoters, low complexity, and other challenging regions*

Multiplex with confidence

  • 24 barcoded adapters available in sets of 8
  • Stringent quality-controlled adapters with <0.05% cross contamination

Automation compatible configuration

  • Plate-friendly reaction volumes and robot-friendly pipetting volumes to accommodate the requirements for liquid handling

 

*Data on file.

Applications
  • Whole Genome Sequencing
  • ChIP-Seq
  • Whole Exome Sequencing
  • Amplicon sequencing
  • Sequencing
  • Targeted Sequencing (Capture)
  • Methyl-Seq
Kit Specifications and Contents / Storage

Kits can be stored for up to 12 months at -20˚C.

Kits include reagents for end repair, ligation and nick repair, and library amplification. PCR-free versions (without library amplification reagents) are also available. Non-barcoded and barcoded (1 – 24) adapter kits are sold separately.

Components

LPK-Ion_Component Chart

Specifications

Spec
Description
Compatible Platform
Ion Personal Genome Machine® (PGM™), Proton™
Library Type
DNA
Starting Material
Fragmented DNA or amplicons
Input Amount
100 ng – 1 µg
Sequencing Applications
Whole Genome Sequencing Whole Exome Sequencing Targeted Sequencing (custom panels) ChIP-Seq Amplicon Sequencing Methyl-Seq
FAQs
What are the input requirements?

The kit provides all of the enzymes and reaction buffers required for constructing libraries from 100 ng – 1 µg of fragmented, double-stranded DNA.

What are the major steps in library construction?

  • End repair: Produce blunt-ended, 5’-phosphorylated fragments
  • Adapter ligation and nick repair: Ligate dsDNA adapters to blunt-ended library fragments and perform nick repair to yield library fragments flanked by adapters
  • Library amplification (optional): Perform PCR to amplify library fragments carrying appropriate adapter sequences on both ends

What are your Covaris® shearing recommendations?

  • If you are performing a cleanup between shearing and end repair, shear in 10 mM Tris-HCl (pH 8 or 8.5) + 1 mMEDTA.
  • If you are not performing a cleanup between shearing and end repair, shear in 10 mM Tris-HCl (pH 8 or 8.5) + 0.1 mMEDTA.
  • Never shear DNA in water.

Are there any safe stopping points in the sample preparation process?

  • After the end repair cleanup DNA can be stored at 4°C for up to 24 hours.
  • After the first post-ligation cleanup resuspend the washed beads in the appropriate volume of 10 mM Tris-HCl (pH 8.0) and store the reactions at 4°C for up to 24 hours.

What are the primer sequences in the Library Amplification Primer Mix?

Primer P1: 5′- CCACTACGCCTCCGCTTTCCTCTCTATG-3’
Primer A: 5′-CCATCTCATCCCTGCGTGTC-3’

What is the proportion of fragmented DNA that is converted to adapter-ligated molecules?

The proportion of fragmented DNA that is converted to adapter-ligated molecules is typically between 5 and 15%. This applies to high-quality DNA and can be significantly lower for DNA of lesser quality, e.g. FFPE samples. Workflows that contain additional bead-based cleanups or size selection prior to library amplification are likely to result in lower yield of adapter-ligated molecules.

 

 

 

How many cleanups should I perform to remove excess unligated adapter and adapter-dimer molecules prior to library amplification or cluster generation?

While a single bead-based cleanup removes most unligated adapter and adapter-dimer, a second bead-based cleanup is recommended to eliminate any remaining adapter species from the library. If size selection is carried out between adapter ligation and library amplification (or clustering), a single post-ligation cleanup with beads (1X) is usually sufficient prior to size selection. If no post-ligation size selection is carried out, two consecutive 1X bead-based cleanups are recommended.

When should I perform size selection protocols?

Size selection requirements vary widely according to specific applications. Depending on preference the double-sided size selection procedures presented in the Library Preparation User Guide may be omitted entirely, modified, or replaced with alternative size selection procedures (such as manual agarose gel electrophoresis, excision and purification or an automated DNA size selection (e.g. Sage Science Pippin Prep™). Size selection may be carried out at alternative points in the workflow, for example:

  • Prior to end repair of fragmented DNA
  • Immediately before library amplification (as outlined in the User Guide)
  • After library amplification

Which polymerase is used for amplification?

KAPA HiFi HotStart is the enzyme provided in the KAPA HiFi HotStart ReadyMix. This is an antibody-based hot-start formulation of KAPA HiFi DNA Polymerase, a novel B-family DNA polymerase engineered for increased processivity and high fidelity.

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

If cycled to completion (NOT RECOMMENDED), a single 50 µl library amplification PCR can produce 8 µg – 10 µg (16 ng – 200 ng/µl) of amplified library. To minimize over-amplification and associated undesired artifacts, the number of amplification cycles should be optimized to produce an amplified library with a concentration in the range of 10 ng – 30 ng/µl (0.5 µg – 1.5 µg of PCR product per 50 µl reaction). Quantification of adapter-ligated libraries prior to library amplification can greatly facilitate the optimization of library amplification parameters, particularly when a library construction workflow is first established. With the KAPA Library Quantification Kit, the amount of template DNA (adapter-ligated molecules) available for library amplification can be determined accurately. Using a simple algorithm (for exponential amplification), the number of amplification cycles needed to achieve a specific yield of amplified library may be predicted theoretically. The actual optimal number of amplification cycles may be 1 – 3 cycles higher, particularly for libraries constructed from FFPE DNA or other challenging samples, or libraries with a broad fragment size distribution.

How do I know if I over-amplified my library?

In library amplification reactions, primers are typically depleted before dNTPs. When DNA synthesis can no longer take place due to substrate depletion, subsequent rounds of DNA denaturation and annealing result in the separation of complementary DNA strands, followed by imperfect annealing to non-complementary partners. This presumably results in the formation of so-called “daisy-chains” or “tangled knots”, comprising large assemblies of improperly annealed, partially double-stranded, heteroduplex DNA. These species migrate slower and are observed as secondary, higher molecular weight peaks during the electrophoretic analysis of amplified libraries.

What are the consequences of over-amplification?

Excessive library amplification can result in unwanted artifacts such as PCR duplicates, chimeric library inserts, amplification bias, and heteroduplex formation. It is generally best to limit the extent of library amplification as much as possible, while ensuring that sufficient material is generated for QC and sequencing.

How do I assess the quality of my library after preparation?

Library size distribution, and the absence of primer dimers and/or over-amplification products, should be confirmed by means of an electrophoretic method. KAPA Library Quantification Kits are recommended for qPCR-based quantification of libraries.

Ordering

Kits include reagents for end repair, ligation and nick repair, and library amplification. PCR-free versions (without library amplification reagents) are also available. Non-barcoded and barcoded (1 – 24) adapter kits are sold separately.

Kit Code
Roche Cat. No
Description
Kit Size
How to buy
KK8300
07961952001
Low throughput
8 libraries
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KK8301
07961979001
Low throughput
48 libraries
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KK8310
07961987001
Low throughput, PCR free
8 libraries
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KK8311
07961995001
Low throughput, PCR free
48 libraries
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KK8330
07962002001
Adapter Kit, non-barcoded
8 libraries
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KK8331
07962029001
Adapter Kit, barcodes 1 - 8
8 x 6 libraries
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KK8332
07962037001
Adapter Kit, barcodes 9 - 16
8 x 6 libraries
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KK8333
07962045001
Adapter Kit, barcodes 17 - 24
8 x 6 libraries
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Accessories

Kit Code
Roche Cat. No
Description
Kit Size
How to buy
KK8000
07983271001
KAPA Pure Beads (5 mL)
5 mL
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KK8001
07983280001
KAPA Pure Beads (30 mL)
30 mL
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KK8002
07983298001
KAPA Pure Beads (60 mL)
60 mL
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