22 Sea Star WGS Library Preps and 2 Redos
DNA Library Prep on 24 Sea Stars (2 Redos) With 2.5 Min Sonication with 500ng, 100ng Going into DNA Prep
Using 1/4th reactions of the KAPA HyperPrep DNA Library Prep Kit
Sonication
- Prepared 500ng of each sample in 80ul of 10mM tris HCL
- Only 22 samples for sonication, libraries 29 and 43 were either already sonicated or already prepped
| Sample | Library Number | 500ng | ul 10mM Tris HCl to 80ul |
|---|---|---|---|
| CPDB 004 | 25 | 14.2 | 65.8 |
| CPAB2 010 | 26 | 19.8 | 60.2 |
| CHSB 008 | 27 | 18.1 | 61.9 |
| CPBP-008 | 28 | 17.7 | 62.3 |
| CPBO 007 | 30 | 21.8 | 58.2 |
| CHSY-007 | 31 | 26.3 | 53.7 |
| CHSB 002 | 32 | 11.0 | 69.0 |
| CHSY 004 | 33 | 12.5 | 67.5 |
| CHSB 010 | 34 | 33.3 | 46.7 |
| CHSY 005 | 35 | 24.3 | 55.7 |
| CPAB 007 | 36 | 14.7 | 65.3 |
| CHSB 007 | 37 | 23.8 | 56.2 |
| CPBP 005 | 38 | 12.5 | 67.5 |
| CPBO 002 | 39 | 20.1 | 59.9 |
| CPBP 003 | 40 | 16.8 | 63.2 |
| CHSB 005 | 41 | 33.1 | 46.9 |
| CPDB 006 | 42 | 16.9 | 63.1 |
| CPOI 001 | 44 | 18.7 | 61.3 |
| CHSB 006 | 45 | 23.7 | 56.3 |
| CHSY 001 | 46 | 27.6 | 52.4 |
| CPDB-008 | 47 | 31.8 | 48.2 |
| CPAB 010 | 48 | 18.7 | 61.3 |
- Ran sonicator for 2.5 minutes at 25% amplitude, 15 seconds on 15 seconds off. Samples were spun down after 1.25 minutes (general sonicator protocol)
- 1X bead clean after sonication, followed protocol
- Samples were re-suspended in 62.5ul 10mM tris HCl, this is 5*12.5, which is the input volume to the kit. I was then able to take 12.5ul from each sonicated sample and that should be ~100ng
Samples 14 and 43 are Redos and have already been sonicated
End Repair and A-tailing
- Prepared end repair and a-tailing master mix on ice:
- ERAT buffer 1.75μl * 25 = 43.75μl
- ERAT enzyme 0.75μl * 25 = 18.75μl
- Made 24 PCR strip tubes each with 12.5μl of 100ng sheared DNA
- Added 2.5μl of ERAT master mix to each sample
- Vortexed and spun down
- Placed samples in thermocyler A-tailing program in JONP login (~ 1 hour)
Adapter Ligation
- Prepared ligation master mix on ice (no vortexing of the ligase):
- ligation buffer 7.5μl * 25 = 187.5μl
- DNA ligase 2.5μl * 25 = 62.5μl
- nuclease free water 1.25μl * 25 = 31.25μl
- Added 11.25ul ligation master mix to each sample.
- Added 1.25ul of planned adapter to each sample (see below). Adapters were added last to minimize adapter-adapter ligation
- Note here that library 30 got adapter 5, when it was supposed to get 6. Library 29 also has adapter 5. This was noticed and library 30 was given a different index pair
| Sample | Library Number | Adapter Number |
|---|---|---|
| CPDB 004 | 25 | 1 |
| CPAB2 010 | 26 | 2 |
| CHSB 008 | 27 | 3 |
| CPBP-008 | 28 | 4 |
| CPBO 007 | 30 | 5 |
| CHSY-007 | 31 | 6 |
| CHSB 002 | 32 | 8 |
| CHSY 004 | 33 | 9 |
| CHSB 010 | 34 | 10 |
| CHSY 005 | 35 | 11 |
| CPAB 007 | 36 | 12 |
| CHSB 007 | 37 | 1 |
| CPBP 005 | 38 | 2 |
| CPBO 002 | 39 | 3 |
| CPBP 003 | 40 | 4 |
| CHSB 005 | 41 | 5 |
| CPDB 006 | 42 | 6 |
| CPOI 001 | 44 | 8 |
| CHSB 006 | 45 | 9 |
| CHSY 001 | 46 | 10 |
| CPDB-008 | 47 | 11 |
| CPAB 010 | 48 | 12 |
| CPBO 008 | 14 | 2 |
| CPBP 009 | 43 | 7 |
- Pipetted all samples up and down with the multichannel and spun down
- Incubated on the shaker at room temp for 1-2 hours at 200rpm
0.8X Cleanup
- Made fresh 80% EtOH
- Took KAPA Pure Beads out of fridge beforehand to warm to room temp
- After incubation at RT, added 22μl of KAPA pure beads to each sample and pipetted up and down at least 10 times to mix beads careful to avoid bubbles
- Placed tubes on shaker at room temp for 15 minutes
- Placed tubes on magnet plate and removed supernatant from tubes when it was fully clear not disturbing the beads
- Added 200μl of 80% EtOH to each tube while still the magnet not disturbing the beads
- Removed supernatant from each tube on the magnet plate without disturbing the beads
- Added 200μl of 80% EtOH to each tube while still the magnet not disturbing the beads
- Removed ALL the supernatant from each tube on the magnet plate without disturbing the beads. Extra EtOH blobs were removed with p20 pipette tips
- Resuspended beads in 6μl 10mM Tris HCl pH 8 and incubated tubes on shaker for 5 minutes
- Placed tubes back onto the magnet stand and removed 5ul supernatant when clear to new labeled PCR strip tubes
Library Amplification
- Made 2 master mixes for the two sets of index pairs
- MM 4, library numbers 25-36 but not 30
- 6.25 KAPA HiFi * 13 = 81.25ul
- 0.75ul 502 20uM primer * 13 = 9.75ul
- 0,75ul 702 20uM primer * 13 = 9.75ul
- MM 5, library numbers 37-48, including redo 43
- 6.25 KAPA HiFi * 13 = 81.25ul
- 0.75ul 503 20uM primer * 13 = 9.75ul
- 0,75ul 703 20uM primer * 13 = 9.75ul
- Vortexed and spun down mixes and kept on ice
- Added 7.5ul of the appropriate mix to the strip tubes with the 5ul sample
- Added 6.25ul KAPA HiFi to libraries 14 and 30
- Added 0.75ul 503 and 0.75ul 703 to 14
- Added 0.75ul 512 and 0.75ul 712 to 30
- Vortexed and spun down samples
- Placed in the thermocyler Genomic PCR 6 program
2 1X Cleanups
- After PCR, added 12.5μl of KAPA pure beads to each sample and pipetted up and down at least 10 times to mix beads careful to avoid bubbles
- Placed tubes on shaker at room temp for 15 minutes
- Placed tubes on magnet plate and removed supernatant from tubes when it was fully clear not disturbing the beads
- Added 200μl of 80% EtOH to each tube while still the magnet not disturbing the beads
- Removed supernatant from each tube on the magnet plate without disturbing the beads
- Added 200μl of 80% EtOH to each tube while still the magnet not disturbing the beads
- Removed ALL the supernatant from each tube on the magnet plate without disturbing the beads. Extra EtOH blobs were removed with p20 pipette tips
- Resuspended beads in 13μl 10mM Tris HCl pH 8 and incubated tubes on shaker for 5 minutes
- Placed tubes back onto the magnet stand and removed 12.5ul supernatant when clear to new labeled PCR strip tubes
- Added 12.5μl of KAPA pure beads to each sample and pipetted up and down at least 10 times to mix beads careful to avoid bubbles
- Placed tubes on shaker at room temp for 15 minutes
- Placed tubes on magnet plate and removed supernatant from tubes when it was fully clear not disturbing the beads
- Added 200μl of 80% EtOH to each tube while still the magnet not disturbing the beads
- Removed supernatant from each tube on the magnet plate without disturbing the beads
- Added 200μl of 80% EtOH to each tube while still the magnet not disturbing the beads
- Removed ALL the supernatant from each tube on the magnet plate without disturbing the beads. Extra EtOH blobs were removed with p20 pipette tips
- Resuspended beads in 16μl 10mM Tris HCl pH 8 and incubated tubes on shaker for 5 minutes
- Placed tubes back onto the magnet stand and removed 15ul supernatant when clear to new labeled PCR strip tubes
QC
- Followed Qubit protocol for BR DNA
| Sample | Library Number | Library concentration ng/ul |
|---|---|---|
| CPDB 004 | 25 | 30.6 |
| CPAB2 010 | 26 | 45.8 |
| CHSB 008 | 27 | 34.8 |
| CPBP-008 | 28 | 42.4 |
| CPBO 007 | 30 | 35.4 |
| CHSY-007 | 31 | 43.6 |
| CHSB 002 | 32 | 37 |
| CHSY 004 | 33 | 20.8 |
| CHSB 010 | 34 | 10.9 |
| CHSY 005 | 35 | 35.6 |
| CPAB 007 | 36 | 32.6 |
| CHSB 007 | 37 | 18.1 |
| CPBP 005 | 38 | 40.4 |
| CPBO 002 | 39 | 41.6 |
| CPBP 003 | 40 | 31.2 |
| CHSB 005 | 41 | 38.4 |
| CPDB 006 | 42 | 24.8 |
| CPOI 001 | 44 | 27 |
| CHSB 006 | 45 | 11.4 |
| CHSY 001 | 46 | 33.8 |
| CPDB-008 | 47 | 42 |
| CPAB 010 | 48 | 36 |
| CPBO 008 | 14 | 2.4 |
| CPBP 009 | 43 | 39.8 |
- D1000 TapeStation, 11 samples, also tapestationing libraries 2 and 50, after their second cleanup to see if the small peak at ~160bp went away
- See report here
- Looks like after 2 bead cleanups at the end, there is no more peak at ~160
- Looks like 34, 37, and 45 need to be re-done
Written on August 2, 2021