Library Preparation for Illumina

It’s complex, but we have it covered.

KAPA DNA Library Preparation Kits for Illumina® 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 and sequence data quality, particularly from low-input and difficult samples such as FFPE and ChIP DNA.

NEW! KAPA Dual-Indexed Adapter Kits are now available. For more information on KAPA Adapter Kits, scroll down to the Ordering section, or download the KAPA Adapter and Bead Calculator.

Download our KAPA  Adapter and Bead Calculator

 

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

Product Highlights

Achieve greater molecular complexity

  • Higher yields of adapter-ligated library translates to higher molecular complexity
  • Fewer cycles of amplification are needed, which results in lower PCR duplication rates
  • PCR-free workflows possible from inputs ≥100 ng*

Reduce amplification bias and improve coverage

  • KAPA HiFi reduces PCR bias*
  • Improves coverage uniformity of GC- and AT-rich regions, promoters, low complexity and other challenging regions*

Create high-quality libraries from challenging samples

  • High-quality whole exome sequencing from libraries prepared using as little as 10 ng human genomic DNA*
  • High success rates with 250 ng FFPE or less*
  • Routine library construction from ≥100 pg ChIP DNA*
KAPA Library Quantification methods (96- and 384-well) format are available for the Sciclone NGS and NGSx workstation (PerkinElmer) and other instruments used in high-throughput NGS sample preparation pipelines.

Automate with pre-validated scripts

  • PerkinElmer Sciclone® NGS and NGSx
  • Beckman Coulter® Biomek™ FXp and FX dual-pod
  • Agilent® Technologies NGS Workstation Option B
  • Eppendorf® epMotion™ 5075 TMX

 

*Data on file.

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

Kits can be stored for up to 18 months at -20˚C.

Kits include reagents for end repair, A-tailing, ligation and library amplification. PCR-free versions (without library amplification reagent) are also available. “With-bead” kits include PEG/NaCl solution for SPRI bead cleanups.
All 96-library kits are automation friendly.

Components

Specifications

Spec
Description
Compatible Platform
Illumina HiSeq, MiSeq, NextSeq and GAIIx
Library Type
DNA
Starting Material
Fragmented DNA or PCR amplicons
Input Amount
100 pg – 5 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 has been validated for library construction from 10 ng – 5 µg of human genomic DNA for whole-genome shotgun sequencing or targeted sequencing. For smaller genomes, or lower complexity samples such as ChIP DNA successful library construction has been achieved from low nanogram to picogram quantities (≥100 pg) of input DNA.

What are the major steps in library construction?

  • End repair: Produce blunt-ended, 5′-phosphorylated fragments
  • A-tailing: dAMP is added to the 3′-ends of blunt-ended dsDNA library fragments
  • Adapter ligation: dsDNA adapters with 3′-dTMP overhangs are ligated to 3′-A-tailed library fragments
  • Library amplification (optional): Employs PCR to amplify library fragments carrying appropriate adapter sequences on both ends

What are your shearing recommendations?

  • If you areperforming a cleanup between shearing and end repair, shear in 10 mM Tris-HCl (pH 8 or 8.5) + 1 mMEDTA.
  • If you are notperforming 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?

The protocol may be paused safely after any of the bead cleanup steps, as described below:

  • After the end repair cleanup, resuspend the washed beads in 20 µL of 1X A-Tailing Buffer without enzyme and store at 4°C for up to 24 hours.
  • After the A-tailing cleanup resuspend the washed beads in 20 µL of 1X Ligation Buffer (without enzyme or adapter) and store the reactions 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 is the proportion of fragmented DNA that is converted to adapter-ligated molecules?

The proportion of fragmented DNA that is successfully converted to adapter-ligated molecules decreases as input is reduced. When starting library construction (end repair) with >100 ng fragmented DNA, 15%–40% of input DNA is typically recovered as adapter-ligated molecules, whereas the recovery typically ranges from 0.5%–15% for libraries constructed from 100 pg – 100 ng DNA. These figures apply to high quality DNA and can be significantly lower for DNA of lower quality and for workflows that contain additional SPRI® cleanups or size selection prior to library amplification.

What adapters can I use with this kit and at what concentration?

KAPA Dual- or Single-Indexed Adapters are recommended for use with KAPA Library Preparation Kits, except for methyl-seq applications. KAPA Library Preparation Kits are also compatible with non-indexed, single-indexed, and dual-indexed adapters that are routinely used in SeqCap EZ, Illumina TruSeq, 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.

Ligation efficiency is robust for adapter-insert molar ratios from 10:1 to 50:1. As a general guideline, an adapter-insert molar ratio of ~10:1 is recommended for libraries constructed from ≥100 ng  of fragmented DNA. Higher adapter-insert molar ratios may be beneficial for low-input and challenging samples.

High Concentration (30 µM) KAPA Single-Indexed Adapter Kits are recommended for library construction from from ≥100 µg inputs, whereas Low Concentration (1.5 µM) KAPA Single-Indexed Adapter Kits are recommended for inputs ≤100 ng. Around 100 ng, the most appropriate concentration depends on library insert size. KAPA Dual-Indexed Adapters may be used for all inputs with the appropriate dilution. For assistance with adapter compatibility and ordering, please visit kapabiosystems.com/support.

Where do I find more information about KAPA Adapters?

Please refer to the KAPA Single- 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.

What QC testing is performed on KAPA Adapters?

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.

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, A-tailing, ligation and library amplification. PCR-free versions (without library amplification reagent) are also available. “With-bead” kits include PEG/NaCl solution for bead-based cleanups.
All 96-library kits are automation friendly.

Kit Code
Roche Cat. No
Description
Kit Size
How to buy
KK8230
07961863001
Low throughput, "with bead"
8 reactions
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KK8231
07961871001
Low throughput, "with bead", PCR-free
8 reactions
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KK8232
07961880001
Low throughput, "with bead"
48 reactions
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KK8233
07961898001
Low throughput, "with bead", PCR-free
48 reactions
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KK8234
07961901001
High throughput, "with bead"
96 reactions
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KK8235
07961910001
High throughput, "with bead", PCR-free
96 reactions
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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
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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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KK8722
08278555702
KAPA Dual-Indexed Adapter Kit, (15 µM)
96 adapters x 20 µl each
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KK8721
08278539001
KAPA Adapter Dilution Buffer (25 mL)
25 mL
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