Whole Genome Sequencing PCR-free Protocol(using TruSeq Library Prep Kit)

*Always make sure beads and Resuspension Buffer warm up to room temperature before use. Mix beads well.

*Keep End Repair Mix, A-tailing Mix, and Ligation Mix on ice. It is also a good idea to spin them down before use.

*Pipette beads slowly

*Take care not to transfer beads when removing supernatant from tubes.

*Briefly centrifuge tubes after mixing with beads.

*Incubation steps occur on rocker.

Fragment DNA

  1. Dilute 1ug of DNA in 100ul total volume of Buffer AE. Transfer to Bioruptor tube.
  2. Use Bioruptor to get ~400bp average insert size (6 cycles of 30 sec. on and 90 sec. off—High setting).
  3. Run surface tension gel using 2ul of sample to ensure desired base pair size. Run additional cycles if necessary.
  4. Transfer DNA to new 1.7ml tube and concentrate to 50ul using SpeedVac. This will take ~23 minutes.

Clean up fragmented DNA

  1. Vortex room temp sample purification beads for at least 1 minute.
  2. Add 80ul of well-mixed beads to each tube. Mix well and incubate tubes at room temperature for 5 minutes.
  3. Place tubes on magnetic stand at room temperature for 8 minutes or until liquid appears clear.
  4. Remove and discard 125ul of supernatant from each tube.
  5. Add 200ul of freshly prepared 80% EtOH to each tube. Incubate for 30 seconds, then remove and discard supernatant.
  6. Repeat Step 5.
  7. Keeping tubes on magnetic stand, let samples air dry at room temperature for 5 minutes.
  8. Keeping tubes on magnetic stand, add 52.5ul Resuspension Bufferto each tube and remove from stand. Mix thoroughly and incubate at room temperature for 2 minutes.
  9. Place tubes on magnetic stand at room temperature for 5 minutes or until liquid appears clear.
  10. Transfer 50ul of supernatant to new tubes.
  11. Proceed immediately to next step.

End Repair and Size Selection

  1. Preheat incubator to 30°C and obtain ice.
  2. Add 10ul Resuspension Buffer to each tube.
  3. Centrifuge End Repair Mix 2.
  4. Add 40ul End Repair Mix 2 to each tube. Return mix to freezer. Mix thoroughly.
  5. Incubate at 30°C for 30 minutes.
  6. Remove from incubator and place tubes on ice until ready for next step.
  7. Remove large DNA fragments:

Combine Sample Purification Beads and PCR grade water in a tube to create a diluted bead mixture of 160ul per 100 ul of end-repaired sample. Determine the volume using the following formulas which include 15% excess for multiple samples.

Diluted Bead Mixture for a 350bp Insert Size:

Formula / i.e. per 12 samples / Your Calculation
Sample Purification Beads / # of samples x 109.25 ul / 1311 ul
PCR grade water / # of samples x 74.75 ul / 897 ul

Diluted Bead Mixture for a 550bp Insert Size:

Formula / i.e. per 12 samples / Your Calculation
Sample Purification Beads / # of samples x 92 ul / 1104 ul
PCR grade water / # of samples x 92 ul / 1104 ul
  1. Add 160ul of diluted bead mixture to each tube. Mix well and incubate tubes at room temp for 5 minutes.
  2. Place tubes on magnetic stand at room temp for 5 minutes or until liquid appears clear.
  3. Transfer 125 ul ofsupernatant two times (~250ul) to new tubes. Discard beads.
  1. Remove small DNA fragments:
  2. Add 30ul of undiluted beads to each tube. Mix well and incubate tubes at room temp for 5 minutes.
  3. Place tubes on magnetic stand at room temp for 5 minutes or until liquid appears clear.
  4. Remove and discard 138ul of supernatant from each tube, twice (~276ul total).
  5. Keeping tubes on magnetic stand, add 200ul freshly prepared 80% EtOH to each tube. Incubate for 30 seconds, then remove and discard supernatant.
  6. Repeat Step D.
  7. Keeping tubes on magnetic stand, let samples air dry at room temp for 5 minutes.
  8. Add 17.5ul Resuspension Buffer to each tube on stand.
  9. Remove tubes from stand and mix thoroughly. Incubate at room temp for 2 minutes.
  10. Place tubes on magnetic stand at room temp for 5 minutes or until liquid appears clear.
  11. Transfer 15ul (or more if possible) of supernatant from each tube to new strip tubes.
  12. Optional: run surface tension gel to ensure smears are in proper size range (~300-500bp)
  13. Proceed to next step or freeze at -20.

Adenylate 3’ Ends

  1. Add 2.5ul Resuspension Buffer to each tube.
  2. Add 12.5ul thawed A-Tailing Mix to each tube and mix thoroughly.
  3. Incubate tubes in thermal cycler at 37°C for 30 minutes, 70°C for 5 minutes, and hold at 4°C. (JOHN thermocycler  Main Folder  SDCRKAD)
  4. Proceed immediately to next step.

Ligate Adapters

  1. Pre-heat incubator to 30°C.
  2. Transfer tube contents to new epitubes.
  3. Add 2.5ul Resuspension Buffer to each tube.
  4. Remove Ligation Mix 2 from freezer and add 2.5ul to each tube. Immediately return Ligation Mix 2 to freezer.
  5. Add 2.5ul of Adapter Index to each tube. Mix thoroughly.
  6. Incubate tubes at 30°C for 10 minutes.
  7. Place tubes on ice until ready for next step.
  8. Add 5ul Stop Ligation Buffer to each tube and mix thoroughly.
  9. Add 42.5ul well-mixed beads to each tube and mix thoroughly. Incubate at room temp for 5 minutes.
  10. Place tubes on magnetic stand at room temp for 5 minutes or until liquid appears clear.
  11. Remove and discard 80ul of supernatant from each tube.
  12. Keeping tubes on magnetic stand, add 200ul freshly prepared 80% EtOH to each tube. Incubate at room temp for 30 seconds, then remove and discard all supernatant from each tube.
  13. Repeat Step 12.
  14. Keeping tubes on magnetic stand, let samples air dry at room temp for 5 minutes.
  15. Keeping tubes on magnetic stand, add 52.5ul Resuspension Buffer.
  16. Remove tubes from stand and mix thoroughly. Incubate at room temp for 2 minutes.
  17. Place tubes on magnetic stand at room temp for 5 minutes or until liquid appears clear.
  18. Transfer 50ul of supernatant to new epitubes.
  19. Add 50ul well-mixed beads to each tube and mix thoroughly. Incubate at room temp for 5 minutes.
  20. Place tubes on magnetic stand and room temp for 5 minutes or until liquid appears clear.
  21. Remove and discard 95ul supernatant from each tube.
  22. Keeping tubes on magnetic stand, add 200ul freshly prepared 80% EtOH. Incubate at room temp for 30 seconds, then remove and discard all supernatant from each tube.
  23. Repeat Step 22.
  24. Keeping tubes on magnetic stand, let samples air dry at room temp for 5 minutes.
  25. Keeping tubes on magnetic stand, add 22.5ul Resuspension Buffer.
  26. Remove tubes from stand and mix thoroughly. Incubate at room temp for 2 minutes.
  27. Place tubes on magnetic stand at room temp for 5 minutes or until liquid appears clear.
  28. Transfer 20ul (or a bit more to text on gel) of the supernatant to new epitubes.
  29. Proceed to next step or freeze at -20.

Validate Library

  1. Picogreen samples to quantify DNA and run gel to verify size of smears.
  2. KAPA qPCR—optional

Normalize and Pool Libraries

  1. Transfer 5ul of each library to new tube.
  2. Normalize concentration of library to 10nM using EBT Buffer (Tris-Cl 10mM, ph8.5 with 0.1% Tween 20) and mix.
  3. Pool samples (5ul of each normalized sample) and send for sequencing (Paired End 100bp).