TITLE: Library Construction Protocol
AUTHORS: Dakota Betz

[DESCRIPTION]
Library construction workflows must be tailored and optimized to accommodate specific experimental
designs, sample characteristics, sequencing applications and equipment. The protocol provided in this document
is generic, and reaction parameters may be adjusted as required to optimize performance, efficiency and
cost-effectiveness.

[BEFORE_START]
Shipping and Storage
The enzymes provided in this kit are temperature sensitive, and appropriate care should be taken during shipping and
storage. KAPA HyperPlus Kits are shipped on dry ice or ice packs, depending upon country of destination. Upon
receipt, immediately store enzymes and reaction buffers at -15°C to -25°C in a constant-temperature freezer. When
stored under these conditions and handled correctly, the kit components will retain full activity until the expiry date
indicated on the kit label.

Handling
Always ensure that KAPA HyperPlus Kit components have been fully thawed and thoroughly mixed before use.
The End Repair & A-Tailing Buffer and Ligation Buffer may contain precipitates when thawed at 2°C to 8°C. These
buffers must be thawed at room temperature and vortexed thoroughly before use. KAPA HiFi HotStart ReadyMix (2X)
contains isostabilizers and may not freeze completely, even when stored at -15°C to -25°C. Nevertheless, always
ensure that the ReadyMix is fully thawed and thoroughly mixed before use. Reaction master mixes prepared from
the enzymes and buffers for fragmentation, end repair and A-tailing, as well as for ligation, are very viscous and
require special attention during pipetting. Keep all enzyme components and master mixes on ice as long as possible
during handling and preparation.

Quality Control
All kit components are subjected to stringent functional quality control, are free of detectable contaminating exoand
endonuclease activities, and meet strict requirements with respect to DNA contamination.

[GUIDELINES]
 In addition to the information in this section, please consult the KAPA NGS Library Preparation Technical
Guide (available on request from Technical Support at sequencing.roche.com/support) for further guidelines
when designing or optimizing your library construction workflow.

The workflow combines enzymatic steps and employs minimal bead-based cleanups, thereby reducing sample
handling and overall library preparation time to 1.5 – 3 hrs. The kit contains all of the enzymes and reaction buffers
required for:
1. enzymatic fragmentation to produce dsDNA fragments;
2. end repair and A-tailing to produce end-repaired,
5'-phosphorylated, 3'-dA-tailed dsDNA fragments;
3. adapter ligation, during which dsDNA adapters with 3'-dTMP overhangs are ligated to 3'-dA-tailed molecules; and
4. library amplification (optional), which employs highfidelity, low-bias PCR to amplify library fragments
carrying appropriate adapter sequences on both ends. 

[STEPS]
SECTION: Enzymatic Fragmentation
1. 
 
Dilute the amount of dsDNA to be used for library construction as follows:
If the DNA preparation does not contain EDTA, dilute in 10 millimolar (mM) Tris-HCl (pH 8.0 – 8.5) in a total of 35 µL 
If the DNA preparation does contain EDTA, dilute in the EDTA-containing buffer in which samples are currently suspended, in a total of 30 µL. To each reaction with 30 µL of EDTA-containing DNA, add 5 µL of diluted Conditioning Solution.


SECTION: Enzymatic Fragmentation
2. Mix by gentle vortexing or pipetting up and down.
SECTION: Enzymatic Fragmentation
3. Assemble each fragmentation reaction on ice by adding the components in this order:


 

SECTION: Enzymatic Fragmentation
4. Vortex gently and spin down briefly. Return the plate/tube(s) to ice. Proceed immediately to the
next step.
SECTION: Enzymatic Fragmentation
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.

 



SECTION: Enzymatic Fragmentation
6. Transfer reactions to ice, and proceed immediately to End Repair and A-tailing (next section).
SECTION: End Repair &amp; A-tailing
7. 
 

SECTION: End Repair &amp; A-tailing
8. In the same plate/tube(s) in which enzymatic fragmentation was performed, assemble each End Repair and A-tailing reaction as follows:


 

SECTION: End Repair &amp; A-tailing
9. Vortex gently and spin down briefly. Return the reaction plate/tube(s) to ice. Proceed immediately
to the next step.
SECTION: End Repair &amp; A-tailing
10. 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).

 

SECTION: End Repair &amp; A-tailing
11. Proceed immediately to Adapter Ligation (next section).

SECTION: Adapter Ligation
12. Dilute adapter stocks to the appropriate concentration, as outlined in Table 4 (see below).
Table 4. Recommended adapter concentrations for libraries constructed from 1 ng – 1 μg input DNA*

 

SECTION: Adapter Ligation
13. In the same plate/tube(s) in which end repair and A-tailing was performed, assemble each adapter
ligation reaction as follows:

 

SECTION: Adapter Ligation
14. Mix thoroughly and centrifuge briefly.
SECTION: Adapter Ligation
15. Incubate at 20 °C for 15 min.

 

SECTION: Adapter Ligation
16. Proceed immediately to Post-ligation Cleanup
SECTION: Post-ligation Cleanup
17. In the same plate/tube(s), perform a 0.8X beadbased cleanup by combining the following: 

 

SECTION: Post-ligation Cleanup
18. Mix thoroughly by vortexing and/or pipetting up and down multiple times.
SECTION: Post-ligation Cleanup
19. Incubate the plate/tube(s) at room temperature for5 min – 15 min to bind DNA to the beads.

SECTION: Post-ligation Cleanup
20. Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
SECTION: Post-ligation Cleanup
21. Carefully remove and discard the supernatant.
SECTION: Post-ligation Cleanup
22. Keeping the plate/tube(s) on the magnet, add 200 µL of 80% ethanol.

SECTION: Post-ligation Cleanup
23. Incubate the plate/tube(s) on the magnet at room temperature for ≥30 s.

SECTION: Post-ligation Cleanup
24. Carefully remove and discard the ethanol.
SECTION: Post-ligation Cleanup
25. Keeping the plate/tube(s) on the magnet, add 200 µLof 80% ethanol.

SECTION: Post-ligation Cleanup
26. Incubate the plate/tube(s) on the magnet at room temperature for ≥30 s.

SECTION: Post-ligation Cleanup
27. Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
SECTION: Post-ligation Cleanup
28. Dry the beads at room temperature for 3 min – 5 min, or until all of the ethanol has evaporated. Caution: over-drying the beads may result in reduced yield.
SECTION: Post-ligation Cleanup
29. Remove the plate/tube(s) from the magnet.
SECTION: Post-ligation Cleanup
30. Resuspend the beads:
• in 25 µL of elution buffer (10 millimolar (mM) Tris-HCl, pH 8.0 – 8.5) to proceed with Library Amplification (step 5), or

• in 55 µLof elution buffer (10 millimolar (mM)Tris-HCl, pH 8.0 – 8.5) to proceed with double-sided
size selection (Appendix 1).
SECTION: Post-ligation Cleanup
31. Incubate the plate/tube(s) at room temperature for 2 minto elute DNA off the beads.

SECTION: Post-ligation Cleanup
32. Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
SECTION: Post-ligation Cleanup
33. Transfer the clear supernatant to a new plate/tube(s):
• to proceed with Library Amplification (step 5), transfer 20 µL of supernatant, or
• to proceed with double-sided size selection (Appendix 1), transfer 50 µL of supernatant.

SECTION: Library Amplificiation
34. 
 

SECTION: Library Amplificiation
35. Assemble each library amplification reaction as follows:

 

SECTION: Library Amplificiation
36. Mix thoroughly and centrifuge briefly.
SECTION: Library Amplificiation
37. Amplify using the following cycling protocol:

 

SECTION: Library Amplificiation
38. Proceed directly to Post-amplification Cleanup
SECTION: Post-amplification Cleanup
39. In the library amplification plate/tube(s), perform a 1X bead-based cleanup by combining the following:

 

SECTION: Post-amplification Cleanup
40. Mix thoroughly by vortexing and/or pipetting up and down multiple times.
SECTION: Post-amplification Cleanup
41. Incubate the plate/tube(s) at room temperature for5 min – 15 min to bind DNA to the beads.

SECTION: Post-amplification Cleanup
42. Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
SECTION: Post-amplification Cleanup
43. Carefully remove and discard the supernatant.
SECTION: Post-amplification Cleanup
44. Keeping the plate/tube(s) on the magnet, add 200 µL of 80% ethanol.

SECTION: Post-amplification Cleanup
45. Incubate the plate/tube(s) on the magnet at room temperature for ≥30 s.

SECTION: Post-amplification Cleanup
46. Carefully remove and discard the ethanol.
SECTION: Post-amplification Cleanup
47. Keeping the plate/tube(s) on the magnet, add 200 µLof 80% ethanol.

SECTION: Post-amplification Cleanup
48. Incubate the plate/tube(s) on the magnet at room temperature for ≥30 s.

SECTION: Post-amplification Cleanup
49. Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
SECTION: Post-amplification Cleanup
50. Keeping the plate/tube(s) on the magnet, add 200 µL of 80% ethanol.

SECTION: Post-amplification Cleanup
51. Incubate the plate/tube(s) on the magnet at room temperature for ≥30 s.

SECTION: Post-amplification Cleanup
52. Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
SECTION: Post-amplification Cleanup
53. Dry the beads at room temperature for3 min – 5 min, or until all of the ethanol has evaporated. Caution:  over-drying the beads may result in reduced yield.

SECTION: Post-amplification Cleanup
54. Remove the plate/tube(s) from the magnet.
SECTION: Post-amplification Cleanup
55. Thoroughly resuspend the beads in an appropriate volume of elution buffer (10 millimolar (mM) Tris-HCl,
pH 8.0 – 8.5) or PCR-grade water.

 

SECTION: Post-amplification Cleanup
56. Incubate the plate/tube(s) at room temperature for 2 min to elute DNA off the beads.

SECTION: Post-amplification Cleanup
57. Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
SECTION: Post-amplification Cleanup
58. Transfer the clear supernatant to a new plate/tube(s) and proceed with size selection (refer  to Appendix 1), library QC, target capture 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.

SECTION: Appendix 1: Size Selection
59. 
 

 

SECTION: Appendix 1: Size Selection
60. Perform the first (0.7X) size cut (to exclude library molecules larger than ~450 bp) by combining the
following:


 

SECTION: Appendix 1: Size Selection
61. Mix thoroughly by vortexing and/or pipetting up and down multiple times.
SECTION: Appendix 1: Size Selection
62. Incubate the plate/tube(s) at room temperature for 5 min– 15 minin to bind library molecules larger than ~450 bp to the beads.
SECTION: Appendix 1: Size Selection
63. Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
SECTION: Appendix 1: Size Selection
64. Carefully transfer ~80 µL of supernatant containing library molecules smaller than ~450 bp to a new plate/tube. It is critical that no beads are transferred with the supernatant. Discard the plate/tube(s) with the beads to which library molecules larger than ~450 bp were bound.
SECTION: Appendix 1: Size Selection
65. Perform the second size cut (0.9X), to retain library molecules >250 bp) by combining the following: 

 

SECTION: Appendix 1: Size Selection
66. Mix thoroughly by vortexing and/or pipetting up and down multiple times.
SECTION: Appendix 1: Size Selection
67. Incubate the plate/tube(s) at room temperature for 5 min – 15 minn to bind library molecules larger than ~250 bp to the beads.
SECTION: Appendix 1: Size Selection
68. Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
SECTION: Appendix 1: Size Selection
69. Carefully remove and discard the supernatant, which contains library molecules smaller than ~250 bp.
SECTION: Appendix 1: Size Selection
70. Keeping the plate/tube(s) on the magnet, add 200 µLof 80% ethanol.

SECTION: Appendix 1: Size Selection
71. Incubate the plate/tube(s) on the magnet at room temperature for ≥30 s.

SECTION: Appendix 1: Size Selection
72. Carefully remove and discard the ethanol.
SECTION: Appendix 1: Size Selection
73. Keeping the plate/tube(s) on the magnet, add 200 µLof 80% ethanol.

SECTION: Appendix 1: Size Selection
74. Incubate the plate/tube(s) on the magnet at room temperature for ≥30 s.

SECTION: Appendix 1: Size Selection
75. Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
SECTION: Appendix 1: Size Selection
76. Dry the beads for 3 min– 5 min at-25 Room temperature, or until all of the ethanol has evaporated. Caution: over-drying the beads may result in reduced yield.

SECTION: Appendix 1: Size Selection
77. Remove the plate/tube(s) from the magnet.
SECTION: Appendix 1: Size Selection
78. Thoroughly resuspend the beads in the required volume of elution buffer (10 millimolar (mM) Tris-HCl,
pH 8.0 – 8.5).
SECTION: Appendix 1: Size Selection
79. Incubate the plate/tube(s) at room temperature for 2 minto elute DNA off the beads.

SECTION: Appendix 1: Size Selection
80. Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
SECTION: Appendix 1: Size Selection
81. Transfer the clear supernatant with size-selected DNA to a new plate/tube(s) and proceed with the
next step in your workflow, or store DNA at 2 °C to 8 °C for 1 – 2 weeks, or at-15 °Cto -25 °C.

SECTION: Appendix 2: Optimization of Fragmentation Parameters
82. 
 

SECTION: Appendix 2: Optimization of Fragmentation Parameters
83. Quantification of Input DNA

The Qubit fluorometer is recommended for the quantification of high-quality DNA, whereas the
KAPA hgDNA Quantification and QC Kit provides both concentration and quality information for FFPE DNA.
SECTION: Appendix 2: Optimization of Fragmentation Parameters
84. Handling of DNA Samples Containing EDTA

 

SECTION: Appendix 2: Optimization of Fragmentation Parameters
85. For example:
• If your DNA samples are in TE buffer, and your input into library construction is 100 ng, dilute 100 ngof each sample into a final volume of 30 µL (i.e., to 3.33 ng/μL ) using TE buffer.

• All samples will now contain the same final EDTA concentration once diluted to 50 µL for fragmentation.
This concentration is: EDTA concentration in TE buffer x (30 μL/50 μL) = 1 mM x (30 μL/50 μL) = 0.6 millimolar (mM)
   
• Make a 2.2-fold dilution of the Conditioning Solution (as per Table 3 on p. 4), and follow the Library
Construction Protocol (second half of step 1).
SECTION: Appendix 2: Optimization of Fragmentation Parameters
86. 
 

SECTION: Appendix 2: Optimization of Fragmentation Parameters
86.1. Set up a series of test reactions with the appropriate amount of input DNA, and different final concentrations
of Conditioning Solution.
SECTION: Appendix 2: Optimization of Fragmentation Parameters
86.2. Include at least one reaction with the same input of control DNA known to be EDTA-free. The control DNA
should preferably be of the same type and quality as the test samples.
SECTION: Appendix 2: Optimization of Fragmentation Parameters
86.3. Fragment the DNA using the appropriate parameters, as outlined in the Library Construction Protocol:
Enzymatic Fragmentation (step 1). Complete the library construction process, and compare library size
distributions for the test and control samples using an electrophoretic system (see Important Parameters:
Evaluating the Success of Library Construction, p. 8).
SECTION: Appendix 2: Optimization of Fragmentation Parameters
86.4. Titrate the final concentration of Conditioning Solution in the reaction until the test samples yield similar
fragmentation profiles as the EDTA-free control sample, or until the desired library size distribution has been
achieved.
SECTION: Appendix 2: Optimization of Fragmentation Parameters
86.5. A two-step strategy may be the best. Start with 3 – 4 test samples covering a broad range of final Conditioning
Solution concentrations, then perform a finer titration over a narrower concentration range.