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31 Sea Star WGS Library Preps

DNA Library Prep on 31 Sea Stars 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
Sample Library number 500ng ul 10mM tris HCL to 80ul
CPAB2 007 49 16.7 63.3
CPAB-003 50 8.1 71.9
CHSB 011 51 31.3 48.8
CPBO-003 52 25.7 54.3
CPDB 007 53 12.6 67.4
Chip 54 6.3 73.8
CPAB 009 55 19.0 61.0
Bash 56 10.9 69.1
CPDB 002 57 8.1 71.9
CPOI 010 58 11.1 68.9
CPDB 010 59 10.3 69.7
CPAB 002 60 15.2 64.8
CHSY 010 61 24.5 55.5
CPBO 005 63 16.5 63.5
CHSY 002 64 23.7 56.3
CPOI 009 65 14.4 65.6
CHSB 004 66 12.2 67.8
CPBP 010 67 9.8 70.2
CPAB2 009 68 19.5 60.5
CPAB 005 69 7.2 72.8
CPDB 005 70 14.7 65.3
CPAB2 006 72 15.8 64.2
CPOI-003 73 16.7 63.3
CPBP 004 74 8.9 71.1
CPAB 008 75 12.4 67.6
CPBP 002 76 15.1 64.9
CPBP 007 77 13.6 66.4
CPDP-003 78 25.5 54.5
CPAB 011 79 25.3 54.7
CPOI 007 80 15.0 65.0
CPAB2 003 81 17.4 62.6
  • 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

End Repair and A-tailing

  • Prepared end repair and a-tailing master mix on ice:
    • ERAT buffer 1.75μl * 33 = 57.75μl
    • ERAT enzyme 0.75μl * 33 = 24.74μl
  • Made 31 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 * 34 = 255μl
    • DNA ligase 2.5μl * 34 = 85μl
    • nuclease free water 1.25μl * 34 = 42.5μl
  • 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
Sample Library number Adapter Index
CPAB2 007 49 1
CPAB-003 50 2
CHSB 011 51 3
CPBO-003 52 4
CPDB 007 53 5
Chip 54 6
CPAB 009 55 7
Bash 56 8
CPDB 002 57 9
CPOI 010 58 10
CPDB 010 59 11
CPAB 002 60 12
CHSY 010 61 1
CPBO 005 63 3
CHSY 002 64 4
CPOI 009 65 5
CHSB 004 66 6
CPBP 010 67 7
CPAB2 009 68 8
CPAB 005 69 9
CPDB 005 70 10
CPAB2 006 72 12
CPOI-003 73 1
CPBP 004 74 2
CPAB 008 75 3
CPBP 002 76 4
CPBP 007 77 5
CPDP-003 78 6
CPAB 011 79 7
CPOI 007 80 8
CPAB2 003 81 9
  • 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 3 master mixes for the three sets of index pairs
  • MM 6, library numbers 49-60
    • 6.25 KAPA HiFi * 13 = 81.25ul
    • 0.75ul 506 20uM primer * 13 = 9.75ul
    • 0,75ul 706 20uM primer * 13 = 9.75ul
  • MM 7, library numbers 61-72 (not including 62 and 71, already prepped)
    • 6.25 KAPA HiFi * 11 = 68.75ul
    • 0.75ul 506 20uM primer * 11 = 8.25ul
    • 0,75ul 706 20uM primer * 11 = 8.25ul
  • MM 11, libraries 73-81
    • 6.25 KAPA HiFi * 10 = 62.5ul
    • 0.75ul 506 20uM primer * 10 = 7.5ul
    • 0,75ul 706 20uM primer * 10 = 7.5ul
  • Vortexed and spun down mixes and kept on ice
  • Added 7.5ul of the appropriate mix to the strip tubes with the 5ul sample
  • Vortexed and spun down samples
  • Placed in the thermocyler Genomic PCR 6 program

1X Cleanup

  • 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 17μl 10mM Tris HCl pH 8 and incubated tubes on shaker for 5 minutes
  • Placed tubes back onto the magnet stand and removed 16ul supernatant when clear to new labeled PCR strip tubes

QC

Sample Library number Qubit Reading (ng/ul)
CPAB2 007 49 42.6
CPAB-003 50 61.8
CHSB 011 51 48
CPBO-003 52 59.6
CPDB 007 53 43.4
Chip 54 68.4
CPAB 009 55 44.8
Bash 56 67.6
CPDB 002 57 58.2
CPOI 010 58 41.6
CPDB 010 59 51.2
CPAB 002 60 40.6
CHSY 010 61 37.2
CPBO 005 63 51
CHSY 002 64 61.4
CPOI 009 65 35.8
CHSB 004 66 35.4
CPBP 010 67 58.4
CPAB2 009 68 63.4
CPAB 005 69 61.8
CPDB 005 70 44
CPAB2 006 72 45.6
CPOI-003 73 34.4
CPBP 004 74 41.4
CPAB 008 75 37.4
CPBP 002 76 43.6
CPBP 007 77 37.6
CPDP-003 78 40.8
CPAB 011 79 41.8
CPOI 007 80 56.2
CPAB2 003 81 43.6
  • Ran the D1000 TapeStation on 4 of the samples
    • See full report here
Written on July 21, 2021