In this protocol

适用说明:本说明书适用于the KAPA HTP Library Preparation Kit (07961901001), 和 the KAPA HTP Library Preparation Kit for PCR-free workflows (07961910001).
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Product Description

KAPA HTP Library Preparation Kit is designed for high- throughput library construction for Illumina sequencing, starting from fragmented, double-stranded DNA (dsDNA). The kit contains all of the enzymes and reaction buffers required for the following steps of library construction:

  1. end repair, to produce blunt-ended, 5'-phosphorylated dsDNA fragments;
  2. A-tailing, during which dAMP is added to the 3'-ends of blunt-ended dsDNA library fragments;
  3. adapter ligation, during which dsDNA adapters with 3'-dTMP overhangs are ligated to 3'-A-tailed library fragments; and
  4. library amplification (optional), which employs high- fidelity, low-bias PCR to amplify library fragments carrying appropriate adapter sequences on both ends.

Efficient, cost-effective reaction cleanups and higher library yields are achieved through implementation of the “with-bead” strategy developed at The Broad Institute of MIT and Harvard and Foundation Medicine.1 The kit includes PEG/NaCl Solution for this purpose. Adapters and beads required for cleanups after end repair and library amplification are not included. However, KAPA Pure Beads and KAPA Adapters are sold separately.

In order to maximize sequence coverage uniformity, it is critical to minimize library amplification bias. KAPA HiFi DNA Polymerase is designed for low-bias, high- fidelity PCR, and is the reagent of choice for NGS library amplification.2,3,4,5 KAPA HTP Library Preparation Kits include KAPA HiFi HotStart ReadyMix (2X), a ready-to- use PCR mix comprising all the components for library amplification—except primers and template. Kits also include Library Amplification Primer Mix (10X), designed for the high-efficiency amplification of Illumina libraries flanked by adapters containing the P5 and P7 flow cell sequences. A kit without the amplification module (KK8235) is available for PCR-free workflows. They may also be combined with KAPA HiFi Real-time Library Amplification Kits, or with KAPA HiFi HotStart Uracil+ ReadyMix Kits for the amplification of bisulfite-converted libraries.

  1. Fisher, S., et al., Genome Biology 12, R1 (2011).
  2. Oyola, S.O., et al., BMC Genomics 13, 1 (2012).
  3. Quail, M.A., et al., Nature Methods 9, 10 (2012).
  4. Quail, M.A., et al., BMC Genomics 13, 341 (2012).
  5. Ross, M.G., et al., Genome Biology 14, R51 (2013).

Product Applications

KAPA HTP Library Preparation Kits are ideally suited for high-throughput NGS library construction workflows that require end repair, A-tailing, adapter ligation and library amplification (optional). The kit is designed for library construction from a wide range of sample types and inputs (10 ng – 5 µg). For small genomes, cell-free/circulating tumor DNA and lower complexity samples such as ChIP DNA, amplicons or cDNA (for RNA-seq), successful library construction has been achieved from lower inputs (~100 pg or more).

The protocol is automation friendly and may be incorporated into workflows for a wide range of NGS applications, including:
● whole-genome, shotgun sequencing
● whole exome or targeted sequencing, using Roche SeqCap EZ, Agilent SureSelect, Illumina TruSeq, IDT xGen Lockdown Probes, or other hybridization capture systems
● ChIP-seq
● RNA-seq (starting with cDNA)
● methyl-seq (in combination with the KAPA HiFi HotStart
Uracil+ ReadyMix for library amplification).

Specific guidelines for the construction of libraries for target capture using the Roche SeqCap EZ system may be found in the Appendix (p 17).

Process Workflow

Library Construction Protocol

  1. Reagent Preparation
    For maximum stability and shelf-life, the enzyme preparations and concentrated reaction buffers for end repair, A-tailing and ligation are supplied separately in KAPA HTP Library Preparation Kits. For a streamlined “with-bead” protocol, a reagent master mix is prepared for each of these enzymatic steps, as outlined in Tables 5, 6, and 7. Additional reagents required for the KAPA HTP Library Preparation protocol are listed in Table 8.

    Master mixes may be constituted with varying proportions of the total final water requirement. In the examples given in the tables below, all of the required water is included in each master mix, allowing the entire reaction mix to be dispensed in a single pipetting step (after cleanups, beads are resuspended directly in master mix for the next enzymatic step). This strategy may be modified for some automated liquid handling systems. For example, master mixes may be prepared with only a portion of the total volume of water, and the rest of the water added directly to the beads, or to beads resuspended in the reaction mix.

    At safe stopping points, some or all of the water and/or reaction buffer may be added to the beads, for storage at 2°C to 8°C for ≤24 hrs. To resume library construction, prepare the master mix with the remaining volume of water (if applicable) and reaction buffer, and the required volume of enzyme. Recommendations on how to formulate master mixes after safe stopping points are provided in Tables 6B and 7B.

    Master mixes for end repair, A-tailing and ligation may be prepared immediately before use, or stored for ≤1 week at 2°C to 8°C, or ≤3 months at -15°C to -25°C. Master mixes stored at -15°C to -25°C are stable through three freeze-thaw cycles.

    The excess of each master mix or reagent required for processing a full batch of 96 samples is given in each of the tables. This is based on current experience with different liquid handling platforms, and varies as a result of the way reagents are utilized by different systems. When processing batches of less than 48 samples, the maximum excess may not be required.

    Table 5. End repair


    Table 6A. A-tailing (uninterrupted protocol)


    Table 6B. A-tailing (with safe stopping point)


    Table 7A. Adapter ligation (uninterrupted protocol)


    Table 7B. Adapter ligation (with safe stopping point)


    Table 8. Volumes of additional reagents required

    *When using automated liquid handling systems with filtered pipette tips, the volume of 80% ethanol used per wash is often reduced to 150 µL to accommodate the maximum working volume of the tip. In such cases, the total volume of 80% ethanol required for per sample is 1.5 mL, or 2.1 mL with the maximum excess, which equates to ~200 mL for 96 samples.

  2. End Repair Reaction Setup
    2.1
    Assemble each end repair reaction in a tube or well of a PCR plate as follows:


    2.2
    Mix and incubate at 20°C for 30 min.
    2.3
    Proceed immediately to End Repair Cleanup (step 3).

  3. End Repair Cleanup
    3.1
    In the same plate/tube(s), perform a 1.7X bead- based cleanup by combining the following:


    3.2
    Mix thoroughly by vortexing and/or pipetting up and down multiple times.
    3.3
    Incubate the plate/tube(s) at room temperature for 5 – 15 min to bind DNA to the beads.
    3.4
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    3.5
    Carefully remove and discard the supernatant.
    3.6
    Keeping the plate/tube(s) on the magnet, add 200 µL of 80% ethanol.
    3.7
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    3.8
    Carefully remove and discard the ethanol.
    3.9
    Keeping the plate/tube(s) on the magnet, add 200 µL of 80% ethanol.
    3.10
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    3.11
    Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
    3.12
    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.
    3.13
    Remove the plate/tube(s) from the magnet.
    SAFE STOPPING POINT   
    Resuspend the beads in 20 μL of A-Tailing Buffer (1X) (Table 6B), cover the reaction and store at 2°C to 8°C for ≤24 hrs. Do not freeze the samples as this will damage the KAPA Pure Beads. When ready, proceed to A-tailing After Safe Stopping Point (step 4B).

  4. A-tailing
    A-tailing is performed either directly after End Repair Cleanup, or after Safe Stopping Point, where beads were resuspended in A-Tailing Buffer (1X) and stored at 2°C to 8°C for ≤24 hrs. Depending on your chosen workflow, proceed with either A-tailing Immediately (step 4A) or A-tailing After Safe Stopping Point (step 4B).

    4A. A-tailing Immediately
    4A.1
    In the plate/tube(s) containing end repaired DNA and beads, assemble the A-tailing reaction as follows:


    4A.2
    Thoroughly resuspend the beads by vortexing and/or pipetting up and down multiple times.
    4A.3
    Incubate the plate/tube(s) at 30°C for 30 min. 4A.4 Proceed immediately to A-tailing Cleanup (step 5).

    4B. A-tailing After Safe Stopping Point
    4B.1
    To resume library preparation, combine the following reagents to perform A-tailing:


    4B.2
    Thoroughly resuspend the beads by vortexing and/or pipetting up and down multiple times.
    4B.3
    Incubate the plate/tube(s) at 30°C for 30 min. 4B.4 Proceed immediately to A-tailing Cleanup (step 5).

  5. A-tailing Cleanup
    5.1
    In the same plate/tube(s), perform a 1.8X bead- based cleanup by combining the following:


    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 bind DNA 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 remove and discard the supernatant.
    5.6
    Keeping the plate/tube(s) on the magnet, add 200 µL of 80% ethanol.
    5.7
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    5.8
    Carefully remove and discard the ethanol.
    5.9
    Keeping the plate/tube(s) on the magnet, add 200 µL of 80% ethanol.
    5.10
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    5.11
    Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
    5.12
    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.
    5.13
    Remove the plate/tube(s) from the magnet.
    SAFE STOPPING POINT 
    Resuspend the beads in 20 μL of Ligation Buffer (1X) (Table 7B), cover the reaction and store at 2°C to 8°C for ≤24 hrs. Do not freeze the samples as this will damage the KAPA Pure Beads. When ready, proceed to Adapter Ligation After Safe Stopping Point (step 6B).

  6. Adapter Ligation
    Adapter ligation is performed either directly after A-tailing Cleanup, or after the Safe Stopping Point, where beads were resuspended in Ligation Buffer (1X) and stored at 2°C to 8°C for ≤24 hrs. Depending on your chosen workflow, proceed with either Adapter Ligation Immediately (step 6A) or Adapter Ligation After Safe Stopping Point (step 6B).

    6A. Adapter Ligation Immediately
    6A.1
    Dilute adapter stock to the appropriate concentration, as outlined in Table 2 (p. 5).
    6A.2
    In the plate/tube(s) containing A-tailed DNA and beads, assemble the adapter ligation reaction as follows:


    6A.3
    Thoroughly resuspend the beads by vortexing and/or pipetting up and down multiple times.
    6A.4
    Incubate at 20°C for 15 min.
    6A.5
    Proceed immediately to 1st Post-ligation Cleanup (step 7).

    6B. Adapter Ligation After Safe Stopping Point
    6B.1
    Dilute adapter stock to the appropriate concentration, as outlined in Table 2 (p. 5).
    6B.2
    To resume library preparation, combine the following reagents to perform adapter ligation:


    6B.3
    Thoroughly resuspend the beads by vortexing and/or pipetting up and down multiple times.
    6B.4
    Incubate at 20°C for 15 min.
    6B.5
    Proceed immediately to 1st Post-ligation Cleanup (step 7).

  7. 1st Post-ligation Cleanup
    Depending on your requirements and chosen workflow, one or two post-ligation cleanups should be performed. Consult Important Parameters: Post-ligation Processing (p. 5) and the KAPA NGS Library Preparation Technical Guide for more information.
    7.1
    In the same plate/tube(s), perform a 1X 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 bind DNA 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 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.
    7.13
    Remove the plate/tube(s) from the magnet.
    7.14
    Thoroughly resuspend the beads in an appropriate volume of elution buffer (10 mM Tris-HCl, pH 8.0 – 8.5), and incubate the plate/tube(s) at room temperature for 2 min to elute DNA off the beads. Recommended volumes are as follows:
    ● If proceeding to 2nd Post-ligation Cleanup
    (step 8), resuspend the beads in 50 µL.
    ● If proceeding to Double-sided Size Selection (step 9), resuspend the beads in 100 µL, and omit the second post-ligation cleanup described below.
    ● For a safe stopping point, resuspend the beads in either 50 µL or 100 µL (as required), and store at 2°C to 8°C for ≤24 hrs.

  8. 2nd Post-ligation Cleanup
    8.1
    In the same plate/tube(s), perform a second 1X bead-based cleanup by combining the following:


    8.2
    Mix thoroughly by vortexing and/or pipetting up and down multiple times.Incubate the plate/tube(s) at room temperature for 5 – 15 min to bind DNA to the beads.
    8.3
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    8.4
    Carefully remove and discard the supernatant.
    8.5
    Keeping the plate/tube(s) on the magnet, add 200 µL of 80% ethanol.
    8.6
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    8.7
    Carefully remove and discard the ethanol.
    8.8
    Keeping the plate/tube(s) on the magnet, add 200 µL of 80% ethanol.
    8.9
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    8.10
    Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
    8.11
    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.
    8.12
    Remove the plate/tube(s) from the magnet.
    8.13
    Thoroughly resuspend the beads in 25 µL of elution buffer (10 mM Tris-HCl, pH 8.0 – 8.5). Consult Important Parameters: Post-ligation Processing (p. 5) for more information on elution volumes.
    8.14
    Incubate the plate/tube(s) at room temperature for 2 min to elute DNA off the beads.
    8.15
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    8.16
    Transfer the clear supernatant to a new plate/ tube(s) and proceed with Library Amplification (step 10), QC, and/or sequencing, as appropriate.

  9. Double-sided Size Selection (250 – 450 bp)
    The double-sided size selection procedure described here is designed for selection of adapter- ligated fragments approximately 250 – 450 bp in length. Please consult the KAPA NGS Library Preparation Technical Guide or contact Technical Support at sequencing.roche.com/support if you wish to select a different range of fragment lengths.
    9.1
    In the same plate/tube(s) used for the first post- ligation cleanup, perform the first size cut (0.6X) by combining the following:


    9.2
    Mix thoroughly by vortexing and/or pipetting up and down multiple times.
    9.3
    Incubate the plate/tube(s) at room temperature for 5 – 15 min to bind library fragments larger than ~450 bp to the beads.
    9.4
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    9.5
    Carefully transfer 155 µL of supernatant containing library fragments smaller than ~450 bp to a new plate/tube(s). It is critical that no beads are transferred with the supernatant.
    9.6
    Discard the plate/tube(s) with beads, to which library fragments larger than ~450 bp are bound.
    9.7
    Perform the second size cut (0.8X) by combining the following:


    9.8
    Mix thoroughly by vortexing and/or pipetting up and down multiple times.
    9.9
    Incubate the plate/tube(s) at room temperature for 5 – 15 min to bind library fragments larger than ~250 bp to the beads.
    9.10
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    9.11
    Carefully remove and discard the supernatant, which contains library fragments smaller than ~250 bp.
    9.12
    Keeping the plate/tube(s) on the magnet, add 200 µL of 80% ethanol.
    9.13
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    9.14
    Carefully remove and discard the ethanol.
    9.15
    Keeping the plate/tube(s) on the magnet, add 200 µL of 80% ethanol.
    9.16
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    9.17
    Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
    9.18
    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.
    9.19
    Remove the plate/tube(s) from the magnet.
    9.20
    Thoroughly resuspend the beads in 25 µL of elution buffer (10 mM Tris-HCl, pH 8.0 – 8.5). Consult Important Parameters: Post-ligation Processing (p. 5) for more information on elution volumes.
    9.21
    Incubate the plate/tube(s) at room temperature for 2 min to elute DNA off the beads.
    9.22
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    9.23
    Transfer the clear supernatant containing size- selected DNA to a new plate/tube(s) and proceed with Library Amplification (step 10), QC, and/or sequencing, as appropriate.

  10. Library Amplification
    Note: please refer to Important Parameters: Library Amplification (p. 6) and the KAPA NGS Technical Guide for more information on optimizing library amplification.
    10.1
    Assemble each library amplification reaction as follows:

    *Or another, suitable 10X library amplification primer mix. The recommended final concentration of each primer in the library amplification reaction is 0.5 – 2 µM. Also refer to Important Parameters: Library Amplification (p. 6).

    10.2
    Mix thoroughly and centrifuge briefly.
    10.3
    Amplify using the following cycling protocol:

    *Optimization of the annealing temperature may be required for non- standard (i.e., other than Illumina TruSeq) adapter/primer combinations.

    10.4
    Proceed directly to Library Amplification Cleanup (step 11).

  11. Library Amplification Cleanup
    11.1
    In the library amplification plate/tube(s), perform a 1X bead-based cleanup by combining the following:


    11.2
    Mix thoroughly by vortexing and/or pipetting up and down multiple times.
    11.3
    Incubate the plate/tube(s) at room temperature for 5 – 15 min to bind DNA to the beads.
    11.4
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    11.5
    Carefully remove and discard the supernatant.
    11.6
    Keeping the plate/tube(s) on the magnet, add 200 µL of 80% ethanol.
    11.7
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    11.8
    Carefully remove and discard the ethanol.
    11.9
    Keeping the plate/tube(s) on the magnet, add 200 µL of 80% ethanol.
    11.10
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    11.11
    Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
    11.12
    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.
    11.13
    Remove the plate/tube(s) from the magnet.
    11.14
    Thoroughly resuspend the beads in an appropriate volume of elution buffer (10 mM Tris-HCl, pH 8.0 – 8.5) or PCR-grade water. Always use PCR-grade water if proceeding to target capture.
    11.15
    Incubate the plate/tube(s) at room temperature for 2 min to elute DNA off the beads.
    11.16
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    11.17
    Transfer the clear supernatant to a new plate/ tube(s) and proceed with library QC, target enrichment, or sequencing, 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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