Preparing ezRAD libraries using the KAPA Biosystems Hyper Prep kit. The full protocol from KAPA Biosystems can be found here.

End repair and A-tailing

Combine the following for each sample in PCR tubes:

reagent volume (µL)
End repair and A-tailing buffer 3.5
End repair and A-tailing enzyme mix 1.5
DNA (digested and bead-cleaned) 25
Total: 30

Mix and spin down briefly, then run on thermocycler:

  • 30 min @ 20°C
  • 30 min @ 65°C
  • Hold @ 4°C

in @ 08:42, out @ 09:42

Adapter ligation

Combine the following in PCR tubes (same tubes that A-tailing was performed in):

reagent volume (µL) MasterMix for 3 rxns (x3.1)
H2O 2.5 7.75
Ligation buffer 15 46.5
DNA Ligase 5 15.5
Adapter (15 µM) 2.5 –
DNA (post A-tailing) 30 –
Total: 55 22.5 to each tube

Add 22.5 µL of water/buffer/ligase mastermix to each tube, then add 2.5 µL of each adapter. Adapters graciously donated by Ingrid from the ToBo lab, from Illumina kit at 15 µM concentration.

library adapter ID adapter sequence
BC 16 CCGTCC(C)
NC 18 GTCCGC(A)
ND 19 GTGAAA(C)

Mix and spin down briefly, then run on thermocycler:

  • 15 min @ 20°C
  • Hold @ 4°C

in @ 09:55, out @ 10:10

Post-ligation bead clean

Bead clean with 0.8:1 vol. beads:DNA.

  1. Add 44 µL Ampure XP beads to 55 µL digested DNA (0.8:1 vol. beads:DNA)
  2. Incubate at room temperature for 5 min.
  3. Place on magnetic stand for 5 min.
  4. Remove supernatant into waste container, leaving ~5 µL behing
  5. Add 200 µL freshly made 80% ethanol (on magnetic stand)
  6. Incubate 30 sec. then remove supernatant to waste container
  7. Add 200 µL feshly made 80% ethanol (on magnetic stand)
  8. Incubate 30 sec. then remove supernatant to waste container
  9. Remove any residual ethanol using pipet
  10. Let dry for 5 min., not longer than 10 min.
  11. Resuspend beads in 27 µL H2O, pipet up and down
  12. Incubate at room temperature for 5 min.
  13. Place on magnetic stand for 5 min.
  14. Transfer 25 µL supernatant to destination tube

Size selection (for ~350-700bp insert)

This is a dual-SPRI bead selection, i.e., removing fragments at both the high and low ends of the distribution. The specific volumes used here represent a “0.7x - 0.5x” (left-side to right-side selection) to select for fragments between ~350 and 700bp.

  1. Add 12.5 µL SPRIselect beads to 25 µL DNA (0.5:1 vol. beads:DNA)
    • This is the “right-side” selection. Fragments >~700bp bind to beads.
  2. Incubate at room temperature for 5 min.
  3. Place on magnetic stand for 5 min.
  4. Transfer 35 µL supernatant to new tube
    • This contains fragments < 700bp, the ones we want to keep.
  5. Add 5 µL SPRIselect beads to kept supernatant.
    • This is the “left-side” selection, and the bead solution:DNA volume ratio is now 0.7:1 because supernatant was already at 0.5:1, and this adds 0.2x vols more bead solution relative to original DNA volume.
  6. Incubate at room temperature for 5 min.
  7. Place on magnetic stand for 5 min.
  8. Remove supernatant into waste container, leaving ~5 µL behing
  9. Add 200 µL freshly made 80% ethanol (on magnetic stand)
  10. Incubate 30 sec. then remove supernatant to waste container
  11. Add 200 µL feshly made 80% ethanol (on magnetic stand)
  12. Incubate 30 sec. then remove supernatant to waste container
  13. Remove any residual ethanol using pipet
  14. Let dry for 5 min., not longer than 10 min.
  15. Resuspend beads in 13 µL 10 mM Tris-HCl, pipet up and down
  16. Incubate at room temperature for 5 min.
  17. Place on magnetic stand for 5 min.
  18. Transfer 11 µL supernatant to destination tube

Nanodrop libraries

library [DNA] (ng/µL) 260:280 260:230
BC 4.4 1.96 9.94
NC 6.8 2.00 4.56
ND 5.6 1.57 2.74

Remaining 10 µL given to Amy Eggers at HIMB Core Lab for library validation (BioAnalyzer and qPCR) and sequencing.