TITLE: Appendix 1: Size Selection
AUTHORS: Dakota Betz

[DESCRIPTION]
Any commonly used size selection technique (e.g., the double-sided size selection described here, or an electrophoretic
method) may be integrated into the KAPA HyperPlus library construction workflow. Size selection should preferably be
carried out after the post-ligation cleanup, or after library amplification. Whether or not size selection is performed, which technique is used, and at what stage of the library construction process it is performed, depends on the nature of the sample, input into library construction, and the sequencing application and read length. For more information on size selection, refer to Important Parameters: Size Selection (p. 7).

[GUIDELINES]
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]
1. 
 

 

2. Perform the first (0.7X) size cut (to exclude library molecules larger than ~450 bp) by combining the
following:


 

3. Mix thoroughly by vortexing and/or pipetting up and down multiple times.
4. Incubate the plate/tube(s) at room temperature for 5 min– 15 minin to bind library molecules larger than ~450 bp to the beads.
5. Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
6. 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.
7. Perform the second size cut (0.9X), to retain library molecules >250 bp) by combining the following: 

 

8. Mix thoroughly by vortexing and/or pipetting up and down multiple times.
9. Incubate the plate/tube(s) at room temperature for 5 min – 15 minn to bind library molecules larger than ~250 bp to the beads.
10. Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
11. Carefully remove and discard the supernatant, which contains library molecules smaller than ~250 bp.
12. Keeping the plate/tube(s) on the magnet, add 200 µLof 80% ethanol.

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

14. Carefully remove and discard the ethanol.
15. Keeping the plate/tube(s) on the magnet, add 200 µLof 80% ethanol.

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

17. Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
18. 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.

19. Remove the plate/tube(s) from the magnet.
20. Thoroughly resuspend the beads in the required volume of elution buffer (10 millimolar (mM) Tris-HCl,
pH 8.0 – 8.5).
21. Incubate the plate/tube(s) at room temperature for 2 minto elute DNA off the beads.

22. Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
23. 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.