In this protocol

适用说明:本说明书适用于KAPA Library Preparation Kits for Ion Torrent (07961952001 与 07961979001), 和 KAPA Library Preparation Kits for Ion Torrent for PCR-free workflows (07961987001 与 07961995001).
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Product Description

This KAPA Library Preparation Kit is designed for the preparation of libraries for sequencing on the Ion Personal Genome Machine (PGM) and Ion Proton semiconductor sequencers. The kit provides all of the enzymes and reaction buffers required for constructing libraries from 100 ng – 1 µg of fragmented, double-stranded DNA via the following steps:

  1. end repair: produce blunt-ended, 5'-phosphorylated fragments.
  2. adapter ligation and nick repair: ligate dsDNA adapters to blunt-ended library fragments and perform nick repair to yield library fragments flanked by adapters.
  3. library amplification (optional): perform PCR to amplify library fragments carrying appropriate adapter sequences on both ends.

The kit provides all of the enzymes and buffers required for library construction and amplification, but does not include adapters or beads. Enzyme formulations and reaction buffers for end repair and ligation and nick repair are supplied in convenient, concentrated formats. KAPA Pure Beads and Ion Torrent Adapters are available separately. These formulations ensure maximum stability of the reagents and high reaction efficiencies while simplifying reaction setup.

In order to maximize sequence coverage uniformity, it is critical that library amplification bias be kept to a minimum. KAPA HiFi DNA Polymerase has been designed for low- bias, high-fidelity PCR, and is the reagent of choice for next generation sequencing (NGS) library amplification.1,2,3 The KAPA Library Preparation Kit for Ion Torrent (KK8300, KK8301) includes KAPA HiFi HotStart ReadyMix (2X) and an adapter-specific primer mix for library amplification. Kits without library amplification components (KK8310 and KK8311) are available for PCR-free workflows.

  1. Oyola, S.O., et al., BMC Genomics 13, 1 (2012).
  2. Quail, M.A., et al., Nature Methods 9, 10 (2012).
  3. Quail, M.A., et al., BMC Genomics 13, 341 (2012).

Product Applications

The KAPA Library Preparation Kit for Ion Torrent Platforms is ideally suited for NGS library construction workflows that involve end repair, adapter ligation and nick repair, and library amplification (optional). The protocol may be adapted for incorporation into workflows for a wide range of NGS applications, including:
● targeted sequencing by solution hybrid selection,
● amplicon sequencing,
● whole-genome shotgun sequencing,
● RNA-seq, and
● ChIP-seq.

Library Preparation Protocol

  1. End Repair Reaction Setup
    1.1
    Assemble each end repair reaction as follows:


    1.2
    Mix and incubate at 20ºC for 30 min.
    1.3
    Proceed immediately to End Repair Cleanup (step 2).

  2. End Repair Cleanup
    2.1
    To each 70 µL end repair reaction, add:


    2.2
    Mix thoroughly by vortexing and/or pipetting up and down multiple times.
    2.3
    Incubate the plate/tube(s) at room temperature for 5 – 15 min to bind DNA to the beads.
    2.4
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    2.5
    Carefully remove and discard the supernatant.
    2.6
    Keeping the plate/tube(s) on the magnet, add 200 µL of 80% ethanol.
    2.7
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    2.8
    Carefully remove and discard the ethanol.
    2.9
    Keeping the plate/tube(s) on the magnet, add 200 µL of 80% ethanol.
    2.10
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    2.11
    Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
    2.12
    Dry the beads at room temperature for 3 – 5 min, or until all of the ethanol has evaporated.
    Caution: over-drying the beads may result in reduced yield.
    2.13
    Remove the plate/tube(s) from the magnet.
    2.14
    Resuspend the beads in 35 μL of elution buffer (10 mM Tris-HCl, pH 8.0 – 8.5) by vortexing thoroughly, or by pipetting up and down multiple times.
    2.15
    Incubate the plate/tube(s) at room temperature for 2 min to elute the DNA off the beads.
    2.16
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    2.17
    Transfer 30 μL supernatant to a new plate/tube(s) and proceed with Adapter Ligation and Nick Repair Reaction Setup (step 3).

    SAFE STOPPING POINT 
    If you are not proceeding immediately to Adapter Ligation and Nick Repair Reaction Setup (step 3), the protocol can be stopped here. Store at 2°C to 8°C or -15°C to -25°C overnight.

  3. Adapter Ligation and Nick Repair Reaction Setup
    3.1
    To each plate/tube(s) containing 30 µL of end-repaired DNA, add:


    3.2
    Mix and incubate the plate/tube(s) at 20°C for 15 min, followed by 65°C for 5 min.
    3.3
    Proceed immediately with Ligation and Nick Repair Cleanup (step 4).

  4. Ligation and Nick Repair Cleanup
    Depending on requirements and chosen workflow, either one or two post-ligation cleanups should be performed. Consult Important Parameters: Post- ligation Processing (p. 4) for more information.

    4A. 1st Post-ligation Cleanup
    4A.1
    To each 70 µL ligation and nick repair reaction, add:


    4A.2
    Mix thoroughly by vortexing and/or pipetting up and down multiple times.
    4A.3
    Incubate the plate/tube(s) at room temperature for 5 – 15 min to bind DNA to the beads.
    4A.4
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    4A.5
    Carefully remove and discard the supernatant. 
    4A.6
    Keeping the plate/tube(s) on the magnet, add 200 µL of 80% ethanol.
    4A.7
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    4A.8
    Carefully remove and discard the ethanol.
    4A.9
    Keeping the plate/tube(s) on the magnet, add 200 µL of 80% ethanol.
    4A.10
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    4A.11
    Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
    4A.12
    Dry the beads at room temperature for 3 – 5 min, or until all of the ethanol has evaporated.
    Caution: over-drying the beads may result in reduced yield.
    4A.13
    Remove the plate/tube(s) from the magnet.
    4A.14
    Thoroughly resuspend the beads in an appropriate volume of 10 mM Tris-HCl (pH 8.0 – 8.5). Incubate the plate/tube(s) at room temperature for 2 min to elute the DNA off the beads. Suggested volumes are as follows:
    ● If proceeding to a second post-ligation cleanup, resuspend the beads in 52 µL, place plate/ tube(s) on the magnet and transfer 50 µL of the supernatant to a new plate/tube(s). Proceed to 2nd Post-ligation Cleanup (step 4B).
    ● If proceeding to double-sided size selection, resuspend the beads in 105 µL, place plate/ tube(s) on the magnet and transfer 100 µL of the supernatant to a new plate/tube(s). Proceed to Double-sided Size Selection (step 5).
    ● If proceeding to amplification or template preparation, resuspend the beads in 25 µL, place plate/tube(s) on the magnet and transfer the clear supernatant to a new plate/tube(s). Proceed with Library Amplification (step 6) or template preparation, as appropriate.

    4B. 2nd Post-ligation Cleanup (optional)
    4B.1
    To each 50 µL of supernatant from the first ligation and nick repair reaction cleanup, add:


    4B.2
    Mix thoroughly by vortexing and/or pipetting up and down multiple times.
    4B.3
    Incubate the plate/tube(s) at room temperature for 5 – 15 min to bind DNA to the beads.
    4B.4
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    4B.5
    Carefully remove and discard the supernatant.
    4B.6
    Keeping the plate/tube(s) on the magnet, add 200 µL of 80% ethanol.
    4B.7
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    4B.8
    Carefully remove and discard the ethanol.
    4B.9
    Keeping the plate/tube(s) on the magnet, add 200 µL of 80% ethanol.
    4B.10
    0 Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    4B.11
    Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
    4B.12
    Dry the beads at room temperature for 3 – 5 min, or until all of the ethanol has evaporated.
    Caution: over-drying the beads may result in reduced yield.
    4B.13
    Thoroughly resuspend the beads in 25 µL of 10 mM Tris-HCl (pH 8.0 – 8.5).
    4B.14
    Incubate the plate/tube(s) at room temperature for 2 min to elute the DNA off the beads.
    4B.15
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    4B.16
    Transfer the clear supernatant to a new plate/ tube(s) and proceed with Library Amplification (step 6) or template preparation, as appropriate.

  5. Double-sided Size Selection (optional)
    5.1
    To 100 µL of DNA in 10 mM Tris-HCl (pH 8.0 – 8.5), add:


    5.2
    Mix thoroughly by vortexing and/or pipetting up and down multiple times.
    5.3
    Incubate the plate/tube(s) at room temperature for 5 – 15 min to allow larger library fragments to bind to the beads.
    5.4
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    5.5
    Carefully transfer the supernatant containing the smaller library fragments to new plate/tube(s), taking care not to transfer any beads with the supernatant.
    5.6
    Discard the old plate/tube(s) containing the larger library fragments.
    5.7
    To the supernatant in the new plate/tube(s) add:


    5.8
    Mix thoroughly by vortexing and/or pipetting up and down multiple times.
    5.9
    Incubate the plate/tube(s) at room temperature for 5 – 15 min to allow the DNA to bind to the beads.
    5.10
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    5.11
    Carefully remove and discard the supernatant. 
    5.12
    Keeping the plate/tube(s) on the magnet, add 200 µL of 80% ethanol.
    5.13
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    5.14
    Carefully remove and discard the ethanol.
    5.15
    Keeping the plate/tube(s) on the magnet, add 200 µL of 80% ethanol.
    5.16
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    5.17
    Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
    5.18
    Dry the beads for 3 – 5 min at room temperature, or until all of the ethanol has evaporated.
    Caution: over-drying the beads may result in reduced yield.
    5.19
    Remove the plate/tube(s) from the magnet. 
    5.20
    Thoroughly resuspend the beads in 25 µL of 10 mM Tris-HCl (pH 8.0 – 8.5).
    5.21
    Incubate the plate/tube(s) at room temperature for 2 min to elute the DNA off the beads.
    5.22
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    5.23
    Transfer the clear supernatant to a new plate/ tube(s) and proceed with Library Amplification (step 6) or template preparation, as appropriate.

  6. Library Amplification Reaction Setup
    Please refer to Important Parameters (p. 3) for more information on optimizing library amplification.
    6.1
    Assemble each library amplification reaction as follows:

    *The recommended final concentration of each primer in the library amplification reaction is 500 nM.

    6.2
    Perform PCR with the following thermocycling parameters:

    *Refer to Table 2 and Figure 1.

    6.3
    Proceed directly to Library Amplification Cleanup (step 7).

  7. Library Amplification Cleanup
    7.1
    In the library amplification plate/tube(s), perform a bead-based cleanup by combining the following:


    7.2
    Mix thoroughly by vortexing and/or pipetting up and down multiple times.
    7.3
    Incubate the plate/tube(s) at room temperature for 5 – 15 min to allow the DNA to bind to the beads.
    7.4
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    7.5
    Carefully remove and discard the supernatant.
    7.6
    Keeping the plate/tube(s) on the magnet, add 200 µL of 80% ethanol.
    7.7
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    7.8
    Carefully remove and discard the ethanol.
    7.9
    Keeping the plate/tube(s) on the magnet, add 200 µL of 80% ethanol.
    7.10
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    7.11
    Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
    7.12
    Dry the beads for 3 – 5 min at room temperature, or until all of the ethanol has evaporated.
    Caution: over-drying the beads may result in reduced yield.
    7.13
    Remove the plate/tube(s) from the magnet.
    7.14
    Resuspend the beads in an appropriate volume of 10 mM Tris-HCl (pH 8.0 – 8.5) and mix thoroughly by vortexing, or by pipetting up and down multiple times.
    7.15
    Incubate the plate/tube(s) at room temperature for 2 min to elute the DNA off the beads.
    7.16
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    7.17
    Transfer the clear supernatant to a new plate/ tube(s) and proceed with library QC and template preparation, as appropriate. Store purified, amplified libraries at 2°C to 8°C for 1 – 2 weeks or at -15°C to -25°C.
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