产品说明书：KAPA Hyper Prep Kit for SureSelectXT Target Enrichment

Library Construction Protocol

Important Notes:
•The following reagents are not supplied in the KAPA Hyper Prep Kit, and must be sourced from general laboratory stocks or third-party suppliers:
Agencourt^® AMPure^® XP reagent (Beckman Coulter, part number A63880, A63881 or A63882) or KAPA Pure Beads (KK8000, KK8001 or KK8002).
Freshly prepared 80% ethanol
Elution buffer (10 mM Tris-HCl, pH 8.0-8.5)
Nuclease-free water (non-DEPC treated).

• Adapters and library amplification primers are provided by Agilent, in your custom SureSelectXT reagent kit.
•Your KAPA Hyper Prep Kit (KK8520) contains all of the KAPA HiFi HotStart ReadyMix needed for pre- and post-capture amplification. Unused ReadyMix may be stored at -20 °C for subsequent use, or at 4 °C (for up to 1 month). KAPA HiFi HotStart ReadyMix stored at -20 °C can withstand 50 freeze-thaw cycles without any impact on performance.
•The Important Parameters section contains background information, as well as important guidelines with respect to DNA input, adapter concentration and the number of pre-capture amplification cycles in Tables 1, 2 and 3. Kindly review the relevant subsections before starting this protocol.

1. Covaris^® shearing of input DNA
   1.1

   Refer to Table 1 on p. 4 to confirm the appropriate amount of input DNA, based on the nature and quality of your DNA samples.
   1.2

   Review the Fragmentation section of Important Parameters to inform decisions about shearing buffer and volume. It is recommended that DNA be sheared in a volume of ~50 µL, in 1X low-EDTA TE buffer (10 mM Tris-HCl [pH 8.0] + 0.1 mM EDTA).
   1.3

   Prepare your Covaris instrument according to the manufacturer’s instructions.
   1.4

   Shear the input DNA to an average size of 150-200 bp, using guidelines provided by Covaris, or a previously validated shearing protocol for your specific input, sample type and quality. Agilent’s recommended parameters are given in the following tables, for instruments with different versions of SonoLab.
   Note: FFPE DNA may require less intense shearing or optimized shearing parameters, depending on the degree of fragmentation/degradation of the DNA in the preparation.
   
   For instruments that use SonoLab 7 or newer:
   
   
   For SonoLab versions earlier than 7:
   
   
   
   1.5

   Transfer the sheared DNA into the tube/ appropriate well of the PCR plate to be used for library construction as quantitatively as possible and proceed to the next section.
   
   
2. End Repair and A-tailing
   2.1

   Assemble each end repair and A-tailing reaction in a tube or well of a PCR plate as follows:
   
   *The buffer and enzyme mix should preferably be pre-mixed and added in a single pipetting step. Premixes are stable for ≤ 24 hours at room temperature, for ≤ 3 days at 4 °C, and for ≤ 4 weeks at -20 °C. When processing multiple samples, prepare a 5-20% excess of master mix, as required for your batch size and manual/automated process.
   
   
   2.2

   Vortex gently and spin down briefly. Return the tube(s)/plate to ice. Proceed immediately to the next step.
   2.3

   Incubate in a thermocycler programmed as outlined below:
   
   *A heated lid is required for this incubation. If possible, set the temperature of the lid at 85 °C, instead of the usual ~105 °C.
   **If proceeding to the adapter ligation reaction setup without any delay, the reaction may be cooled to 20 °C instead of 4 °C.
   
   
   2.4

   Proceed immediately to the next step.
   
   
3. Adapter Ligation
   3.1

   Dilute the SureSelectXT Adaptor Oligo Mix (supplied by Agilent) to the appropriate concentration, as outlined in Table 2 on p. 5.
   3.2

   In the same tube(s)/plate in which end repair and A-tailing was performed, assemble each adapter ligation reaction as outlined in the table below:
   
   *From your SureSelectXT reagent kit, supplied by Agilent. For high- input (500 ng-1 µg) libraries, the volume of Adaptor Oligo Mix may be increased to a maximum of 10 µL, and the volume of water reduced accordingly.
   **The buffer, ligase enzyme and water should preferably be premixed and added in a single pipetting step. Premixes are stable for ≤ 24 hours at room temperature, for ≤ 3 days at 4 °C, and for ≤ 4 weeks at -20 °C. When processing multiple samples, prepare a 5-20% excess of master mix, as required for your batch size and manual/automated process.
   

   
   
   3.3

   Mix thoroughly and centrifuge briefly.
   3.4

   Incubate at 20 °C for 15 min. Do not use a heated lid for this incubation. 
   Note: To achieve higher conversion rates and library yields, particularly for low-input samples, consider increasing the ligation time – to a maximum of 4 h at 20 °C, or overnight at a temperature in the range of 4-16 °C. Please note that longer ligation times may lead to increased levels of adapter-dimer. Adapter concentrations may have to be optimized if ligation times are extended significantly.
   
   3.5

   Proceed immediately to the next step.
   
   
4. Post-ligation Cleanup
   4.1

   In the same tube(s)/plate, perform a 0.8X bead cleanup by combining the following:
   
   *Agencourt^® AMPure^® XP reagent or KAPA Pure Beads
   
   
   4.2

   Mix thoroughly by vortexing and/or pipetting up and down multiple times.
   4.3

   Incubate the tube(s)/plate at room temperature for 10 min to bind DNA to the beads.
   4.4

   Place the tube(s)/plate on a magnet to capture the beads. Incubate until the liquid is clear.
   4.5

   Carefully remove and discard the supernatant.
   4.6

   Keeping the tube(s)/plate on the magnet, add 200 µL of 80% ethanol.
   4.7

   Incubate the tube(s)/plate on the magnet at room temperature for ≥ 30 sec.
   4.8

   Carefully remove and discard the ethanol.
   4.9

   Keeping the tube(s)/plate on the magnet, add 200 µL of 80% ethanol.
   4.10

   Incubate the tube(s)/plate on the magnet at room temperature for ≥ 30 sec.
   4.11

   Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
   4.12

   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 dramatic yield loss.
   4.13

   Remove the tube(s)/plate from the magnet.
   4.14

   Resuspend the beads:
   •in 25 µL of elution buffer (10 mM Tris-HCl, pH 8.0-8.5) or nuclease-free water to proceed with pre-capture library amplification (Section 5).
   •in 55 µL of elution buffer (10 mM Tris-HCl, - pH 8.0-8.5) or nuclease-free water to proceed with an additional post-ligation cleanup (transfer 50 µL and repeat this section from step 4.1, with 50 µL of paramagnetic beads).
   4.15

   Incubate the tube(s)/plate at room temperature for 2 min to elute DNA off the beads.
   4.16

   Place the tube(s)/plate on a magnet to capture the beads. Incubate until the liquid is clear.
   4.17

   Transfer 20 µL of the clear supernatant to a new tube/plate and proceed with pre-capture library amplification (Section 5).
   
   
5. Pre-capture Library Amplification
   5.1

   Assemble each library amplification reaction in a PCR tube or well of a PCR plate as follows:
   
   *From your SureSelectXT reagent kit, supplied by Agilent.
   
   
   5.2

   Mix thoroughly and centrifuge briefly.
   5.3

   Amplify using the cycling protocol outlined in the table below.
   Note: The number of amplification cycles depends on the quality of the DNA used for library construction, the actual input into the end repair and A-tailing reaction, the efficiency of library construction and the amount of each library needed for capture. For the SureSelectXT target enrichment workflow, 750 ng of each library is recommended for hybridization (but 500 ng-1 µg can be accommodated). Use the cycle numbers in Table 3 on p. 7 as a starting point. If the yield from the pre-capture amplification reaction did not exceed 500 ng, the purified library may be re- amplified (for ≥ 2 cycles) using this same protocol, or library construction may be repeated with a higher number of pre-capture amplification cycles. 
   
   
   
   5.4

   Proceed directly to Step 6: Post-amplification Cleanup.
   
   
6. Post-amplification Cleanup
   6.1

   In the library amplification tube(s)/plate perform a 1X bead cleanup by combining the following:
   
   *Agencourt® AMPure® XP reagent or KAPA Pure Beads
   
   
   6.2

   Mix thoroughly by vortexing and/or pipetting up and down multiple times.
   6.3

   Incubate the tube(s)/plate at room temperature for 10 min to bind DNA to the beads.
   6.4

   Place the tube(s)/plate on a magnet to capture the beads. Incubate until the liquid is clear.
   6.5

   Carefully remove and discard the supernatant.
   6.6

   Keeping the tube(s)/plate on the magnet, add 200 µL of 80% ethanol.
   6.7

   Incubate the tube(s)/plate on the magnet at room temperature for ≥ 30 sec.
   6.8

   Carefully remove and discard the ethanol.
   6.9

   Keeping the tube(s)/plate on the magnet, add 200 µL of 80% ethanol.
   6.10

   Incubate the tube(s)/plate on the magnet at room temperature for ≥ 30 sec.
   6.11

   Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
   6.12

   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 dramatic yield loss.
   6.13

   Remove the tube(s)/plate from the magnet.
   6.14

   Thoroughly resuspend the beads in 30 µL of non- DEPC treated nuclease-free water.
   6.15

   Incubate the tube(s)/plate at room temperature for 2 min to elute DNA off the beads.
   6.16

   Place the tube(s)/plate on a magnet to capture the beads. Incubate until the liquid is clear.
   6.17

   Transfer the clear supernatant to a new tube/plate and proceed with library QC.
   6.18

   Determine the concentration and fragment size distribution of pre-capture libraries. Please refer to Important Parameters: Evaluating the Success of Library Construction (p. 7-8) for guidelines.
   Purified, pre-capture libraries may be stored overnight at 4 °C, or up to 6 months at -20 °C before proceeding with SureSelectXT hybridization.
   
   
7. Target Enrichment
   Please refer to the SureSelectXT target enrichment protocol supplied by Agilent for subsequent steps in the workflow, i.e.,
   •SureSelectXT hybridization
   •Capture and washing of hybridization reactions
   
   
8. On-bead Post-capture Amplification
   One 50 µL post-capture library amplification reaction is performed per sample. As template, ~50% of the washed, resuspended streptavidin beads retaining the SureSelectXT captured DNA is used. The remaining 50% of the bead slurry is stored at -20 °C.
   8.1

   Assemble each post-capture library amplification reaction in a PCR tube or well of a PCR plate as described on the next page:
   
   *From your SureSelectXT reagent kit, supplied by Agilent.
   
   
   8.2

   Mix thoroughly and centrifuge briefly.
   8.3

   Into each tube/well with PCR master mix, add 5 µL of the appropriate indexing PCR primer (SureSelect 8 bp Indexes A01 to H12, provided in your Agilent reagent kit; in white-capped tubes or the blue plate). Add only one of the 16 or 96 different indexing primers to each reaction.
   8.4

   Mix each SureSelectXT captured library (generated in Section 7; 30 µL DNA-retaining streptavidin bead slurry per sample) to ensure a homogeneous suspension.
   8.5

   Transfer 14 µL of streptavidin bead suspension to each tube/well containing PCR master mix and indexing primer, for a total reaction volume of 50 µL.
   Note: Make sure that each SureSelectXT captured library is combined with the appropriate indexing primer.
   8.6

   Mix thoroughly but do not centrifuge.
   8.7

   Amplify using the cycling protocol outlined in the table below.
   
   
   Suggested PCR cycles for post-capture PCR:
   
   
   
   8.8

   Proceed directly to Step 9: Final Cleanup.
   
   
9. Final Cleanup
   9.1

   In the library amplification tube(s)/plate perform a 1X SPRI® cleanup by combining the following:
   
   *Agencourt^® AMPure^® XP reagent or KAPA Pure Beads
   
   
   9.2

   Mix thoroughly by vortexing and/or pipetting up and down multiple times.
   9.3

   Incubate the tube(s)/plate at room temperature for 10 min to bind DNA to the beads.
   9.4

   Place the tube(s)/plate on a magnet to capture the beads. Incubate until the liquid is clear.
   9.5

   Carefully remove and discard the supernatant.
   9.6

   Keeping the tube(s)/plate on the magnet, add 200 µL of 80% ethanol.
   9.7

   Incubate the tube(s)/plate on the magnet at room temperature for ≥ 30 sec.
   9.8

   Carefully remove and discard the ethanol.
   9.9

   Keeping the tube(s)/plate on the magnet, add 200 µL of 80% ethanol.
   9.10

   Incubate the tube(s)/plate on the magnet at room temperature for ≥ 30 sec.
   9.11

   Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
   9.12

   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 dramatic yield loss.
   9.13

   Remove the tube(s)/plate from the magnet.
   9.14

   Thoroughly resuspend the beads in 30 µL of elution buffer (10 mM Tris-HCl, pH 8.0-8.5) or nuclease-free water.Note: Elution buffer is preferred for long-term storage.
   9.15

   Incubate the tube(s)/plate at room temperature for 2 min to elute DNA off the beads.
   9.16

   Place the tube(s)/plate on a magnet to capture the beads. Incubate until the liquid is clear.
   9.17

   Transfer the clear supernatant to a new tube/plate and proceed with library QC.
   9.18

   Determine the concentration and fragment size distribution of post-capture libraries. Please refer to Important Parameters: Evaluating the Success of Library Construction (p. 7-8) for guidelines.
   9.19

   Purified, post-capture libraries may be stored overnight at 4 °C, or up to 6 months at -20 °C before proceeding with sequencing.
   9.20

   Pool post-capture libraries in an equimolar fashion, and adjust the final volume of the pool for multiplexed sequencing on the Illumina^® platform. Please refer to your SureSelectXT protocol for an example.