Illumina Library Prep

KAPA HyperPlus kit

Kit KR 1145

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Manual

0. Prepare DNA

Removal of EDTA from DNA samples prior to fragmentation is recommended to ensure consistent results. This may be achieved by means of a 3X bead based cleanup with KAPA cleanup beads. Please refer to the relevant Technical Data Sheet (KR1705 or KR1245) for a detailed DNA cleanup protocol.

For optimal fragmentation results, elute DNA in Elution Buffer (10 mM Tris-HCl, pH 8.0 - 8.5) after the cleanup.

KAPA cleanup bead protocol

1. Fragmentation

• 1.1 Dilute 250 ng of dsDNA in Elution Buffer (10 mM Tris-HCl, pH 8.0 - 8.5) to reach a total volume of 11.7 µL (1/3 rx)
  • only do this if samples do not contain EDTA. Otherwise see original protocol for instructions to clean DNA or use Conditioning Solution.
• 1.2 Mix by gentle vortexing or pipetting up and down
• 1.3 Assemble the fragmentation reaction on ice by adding the following components in order:

Component	Volume	1/3 Volume
250ng DNA	35 µL	11.7 µL
KAPA Frag Buffer(10X)*	5 µL	1.6µL
KAPA Frag Enzyme*	10 µL	3.4 µL
———————-	———	———–
Total Volume	50 µL	16.7 µL

OPTIONAL: The KAPA Frag Buffer and Enzyme may be pre-mixed and kept on ice prior to reaction setup, and dispensed as a single solution.

• PREP: get the End Repair & A-Tailing Buffer out of the freezer to begin melting for next step.

• 1.4 Vortex gently and spin down briefly. Return the plate/tue to ice and proceed immediately to the next step.

• 1.5 Incubate in a thermocycler, pre-cooled to 4°C and programmed as outlined below. A heated lid is not required for this step. If used, set the temperature of the heated lid to ≤50°C.

Step	Temp	Time
Pre-cool block	4°C	N/A
Fragmentation	37°C	7:00*
HOLD	4°C	∞

Fragmentation time was previously optimized for 500bp fragments starting from the optimization table below

Mode Fragment Length	Incubation time at 37°C	Optimization range
600 bp	5 min	3- 10 min
350 bp	10 min	5- 20 min
200 bp	20 min	10- 25 min
150 bp	30 min	20- 40 min

• 1.6 Transfer reactions to ice, and proceed immediately to End Repair and A-tailing (step 2).

2. End Repair and A-tailing

• 2.1 In the same plate/tube(s) in which enzymatic fragmentation was performed, assemble each End Repair and A-tailing reaction as follows:

Component	Volume	1/3 Volume
Fragmented, double-stranded DNA	50 µL	16.7 µL
End Repair & A-Tailing Buffer*	7 µL	2.43 µL
HyperPlus ERAT Enzyme Mix*	3 µL	1 µL
——————–	——	——-
Total Volume	60 µL	20 µL

OPTIONAL: Pre-mix End Repair & A-Tailing Buffer and HyperPlus ERAT Enzyme Mix and keep on ice prior to reaction setup, and dispense as a single solution.

• 2.2 Vortex gently and spin down briefly. Return the reaction plate/tube(s) 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 step. If possible, set the temperature of the heated lid to ~85°C (instead of the usual 105°C).

Step	Temp	Time
End repair and A-tailing	65°C	30 min
HOLD	4°C	∞

• PREP: Thaw reagents of the adapter ligation and pre-make the master mix.

• 2.4 Proceed immediately to Adapter Ligation (step 3).

3. Adapter Ligation

• 3.1 Dilute adapter stocks to the appropriate concentration, as outlined in Table 4 (p. 5) of the manual.

We added 250ng of DNA so we want an Adapter:insert molar ratio of 40:1. Since we are using 1/3 reaction volumes we combined the water and adapter stock volume to add 5 µL and calculated that we need a concentration of [6 µM]. For a more detailed explanation on this see the Adapter Concentration Calculations page.

• 3.2 In the same plate/tube(s) in which end repair andA-tailing was performed, assemble each adapter ligation reaction as follows:

Component	Volume	1/3 Volume	Notes
End repair and A-tailing reaction product	60 µL	20 µL
Adapter stock [6 µM]	5 µL	5 µL
PCR grade water*	5 µL	- µL	Already included in the adapter stock
Ligation Buffer*	30 µL	10 µL
DNA Ligase*	10 µL	3.3 µL
——————–	——	——	——
Total Volume	110 µL	38.3 µL

OPTIONAL: *The water, buffer and ligase enzyme should preferably be premixed and added in a single pipetting step. Premixes are stable for ≤24 hrs at room temperature, for ≤3 days at 2°C to 8°C, and for ≤4 weeks at -15°C to -25°C.

• PREP: Acclimate Kapa beads to RT and make 80% ethanol for the bead cleanup

• 3.3 Mix thoroughly and centrifuge briefly.

• 3.4 Incubate at 20°C for 15 min.

• 3.5 Proceed immediately to Post-ligation Cleanup (step 4).

4. Post Ligation Cleanup

IMPORTANT- Kapa beads should be at RT and well mixed using the vortexer

• PREP: Thaw KAPA HiFi HotStart ReadyMix (2X) of the next step.

• PREP: Warm elution buffer to 37°C

• 4.1 In the same plate/tube(s), perform a 0.8X beadbased cleanup by combining the following:

Component	Volume	1/3 Volume	Notes
Adapter ligation reaction product	110 µL	38.3 µL
KAPA cleanup beads (0.8X)	88 µL	30.64 µL
——————–	——	——	——
Total Volume	198 µL	68.9 µL

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

• 4.3 Incubate the plate/tube(s) at room temperature for 15 min or less to bind DNA to the beads.

• 4.4 Place the plate/tube(s) 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 plate/tube(s) on the magnet, add 100 µL of 80% ethanol.

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

• 4.8 Carefully remove and discard the ethanol.

• 4.9 Keeping the plate/tube(s) on the magnet, add 100 µL of 80% ethanol.

• 4.10 Incubate the plate/tube(s) 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 reduced yield.

• 4.13 Remove the plate/tube(s) from the magnet.

• 4.14 Resuspend the beads in 25 µL of elution buffer (10 mM Tris-HCl, pH 8.0 – 8.5) previously warmed to 37°C

• 4.15 Incubate the plate/tube(s) at room temperature for 5 min to elute DNA off the beads.

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

• 4.17 Transfer 20 µL of supernatant to a new plate/tube(s) to proceed with Library Amplification (step 5)

5. Library Amplification

The KAPA Library Amplification Primer Mix (10X) is designed to eliminate or delay primer depletion during library amplification reactions performed with KAPA HiFi HotStart ReadyMix. The primer mix is suitable for the amplification of all Illumina libraries flanked by the P5 and P7 flow cell sequences. Primers are supplied at a 10X concentration of 20 μM each, and have been formulated as described below. User-supplied primer mixes may be used in combination with incomplete or custom adapters. Please contact Technical Support at sequencing.roche.com/support for guidelines on the formulation of user-supplied library amplification primers.

• To achieve the highest amplification efficiency and avoid primer depletion, it is critical to use an optimal concentration of high-quality primers. Primers should be used at a final concentration of 0.5 – 4 μM each. For libraries constructed from ≥100 ng input DNA, a final concentration of at least 2 μM of each primer is recommended.

• Library amplification primers should be HPLC-purified and modified to include a phosphorothioate bond at the 3’-terminal of each primer (to prevent degradation by the strong proofreading activity of KAPA HiFi HotStart). Always store and dilute primers in a buffered solution (e.g., 10mM Tris-HCl, pH8.0–8.5), and limit the number of freeze-thaw cycles. To achieve the latter, store primers at 2°C to 8°C for short-term use, or as single-use aliquots at -15°C to -25°C.

• 5.1 Assemble each library amplification reaction as follows:

Component	Volume	1/3 Volume	Notes
KAPA HiFi HotStart ReadyMix (2X)	25 µL	8.3 µL
Library Amplification Primer Mix (10X)*	5 µL	1.7 µL	if using custon Primers i5/i7 they should be at [20µM]
Adapter-ligated library	20 µL	6.7 µL
——————–	——	——	——
Total Volume	50 µL	16.7 µL

** NOTE: keep Adapter ligated library extra just in case **

• 5.2 Mix thoroughly and centrifuge briefly.
• 5.3 Amplify using the following cycling protocol:

Step	Temp	Time	Cycles
Initial Denaturation	98°C	45 sec	1
Denaturation	98°C	15 sec
Annealing	60°C	30 sec	8X
Extension	72°C	30 sec
Final Extension	72°C	1 min	1
HOLD	4°C	∞	1

• 5.4 Proceed directly to Post-amplification Cleanup (step 6).

6. Post-ligation Cleanup

IMPORTANT- Only do this the first time and check your library. Otherwise we will skip and go directly to the size selection step.

• 6.1 In the same plate/tube(s), perform a 1X beadbased cleanup by combining the following:

Component	Volume	1/3 Volume	Notes
Library amplification reaction product	50 µL	16.7 µL
KAPA cleanup beads (1X)	50 µL	16.7 µL
——————–	——	——	——
Total Volume	100 µL	33.4 µL

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

• 6.3 Incubate the plate/tube(s) at room temperature for 15 min or less to bind DNA to the beads.

• 6.4 Place the plate/tube(s) 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 plate/tube(s) on the magnet, add 100 µL of 80% ethanol.

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

• 6.8 Carefully remove and discard the ethanol.

• 6.9 Keeping the plate/tube(s) on the magnet, add 100 µL of 80% ethanol.

• 6.10 Incubate the plate/tube(s) 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 reduced yield.

• 6.13 Remove the plate/tube(s) from the magnet.

• 6.14 Resuspend the beads in 25 µL of elution buffer (10 mM Tris-HCl, pH 8.0 – 8.5) previously warmed to 37°C

• 6.15 Incubate the plate/tube(s) at room temperature for 5 min to elute DNA off the beads.

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

• 6.17 Transfer clear supernatant to a new plate/tube(s) and proceed with size selection (step 7)

7. Size Selection

*The original protocol calls for a size selection using 0.7X and 0.9X size selection (~ 250bp - 400bp). We have tested

7.a Uppercut

We are first going to remove any fragments larger than > 600 bp

• 7.a.1 First, add 13.3 µL of molecular H2O to increase the final volume after the PCR from 16.7 µL to 30 µL.

• 7.a.2 In the same plate/tube(s), perform a 0.56X beadbased cleanup by combining the following:

Component	Volume	1/3 Volume	Notes
Library amplification reaction product + H20	50 µL	30 µL
KAPA cleanup beads (0.56X)	28 µL	16.8 µL
——————–	——	——	——
Total Volume	78 µL	46.8 µL

• 7.a.3 Mix thoroughly by vortexing and/or pipetting up and down multiple times.

• 7.a.4 Incubate the plate/tube(s) at room temperature for 15 min or less to bind DNA to the beads.

• 7.a.5 Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.

• 7.a.6 Carefully remove and KEEP THE SUPERNATANT and discard the beads

7.b Lowercut

We are now going to retain fragments > ~300 bp

• 7.b.1 In the same plate/tube(s), perform a 0.72X beadbased cleanup by combining the following:

Component	Volume	1/3 Volume	Notes
Uppercut supernatant (Amplification product)	50 µL	46.8 µL
KAPA cleanup beads (0.72X)	36 µL	21.6 µL	calculated from previous volume of 30 µL
——————–	——	——	——
Total Volume	86 µL	68.4 µL

• 7.b.2 Mix thoroughly by vortexing and/or pipetting up and down multiple times.

• 7.b.3 Incubate the plate/tube(s) at room temperature for 15 min or less to bind DNA to the beads.

• 7.b.4 Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.

• 7.b.5 Carefully remove and discard the supernatant.

• 7.b.6 Keeping the plate/tube(s) on the magnet, add 100 µL of 80% ethanol.

• 7.b.7 Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.

• 7.b.8 Carefully remove and discard the ethanol.

• 7.b.9 Keeping the plate/tube(s) on the magnet, add 100 µL of 80% ethanol.

• 7.b.10 Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.

• 7.b.11 Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.

• 7.b.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.b.13 Remove the plate/tube(s) from the magnet.

• 7.b.14 Resuspend the beads in 22 µL of elution buffer (10 mM Tris-HCl, pH 8.0 – 8.5) previously warmed to 37°C

• 7.b.15 Incubate the plate/tube(s) at room temperature for 5 min to elute DNA off the beads.

• 7.b.16 Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.

• 7.b.17 Transfer 20 µL of clear supernatant (Amplified Library) to a new plate/tube(s)

Congratulations! You are done! Now run your QC steps (e.g., Tapestation, Qubit, RT-PCR) prior to sending for sequencing.