DNA Library Prep for NB Adult Oyster EecSeq - 8 Samples

Initial bead cleanup and library prep were performed on Oct. 15, 2019.

Using the KAPA HyperPrep DNA Library Prep Kit on 8 DNA samples in 10 mM Tris HCl pH 8 containing 500 ng from the first 50 samples of the NB adult oyster experiment.

Previously, all samples were sonicated to 150 bp following the QSonica protocol- instructions can be found here.

A 1.8X bead cleanup was performed to concentrate 500 ng of DNA in 25 μl before library prep:

• Made fresh 80% EtOH
• Took KAPA Pure Beads out of fridge beforehand to warm to room temp
• Vortex and spin down DNA samples
• Added 90 μl (1.8 x 50 μl) of KAPA Pure Beads to each sample and pipette up and down 10 times to mix (avoid bubbles)
• Placed tubes on shaker at room temp for 15 minutes - shaker set to 200 rpm’s
• After 15 minute incubation, placed tubes on magnet plate and removed supernatant from tubes when it was fully clear not disturbing the beads
• Added 200 μl 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 200 μl 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 25 μl of 10 mM Tris HCl pH 8 and incubated tubes on shaker at room temp for 5 minutes
• Placed tubes on magnet plate and transferred supernatant when clear to new labeled PCR strip tubes

End Repair and A-tailing

• Prepared end repair and a-tailing master mix:
  • ERAT buffer: 3.5 μl * 9 = 31.5 μl
  • ERAT enzyme: 1.5 μl * 9 = 13.5 μl
• In the 8 PCR strip tubes containing the 25 μl of 500 ng sheared DNA, added 5 μl of ERAT master mix
• Vortexed and spun down
• Placed samples in thermocycler A-tailing program in JONP login - program runs for ~ 1 hour

Adapter Ligation

Working adapter stocks 1-7, and 9-12 were diluted on Oct. 10, 2019; working adapter stock 8 was diluted on Oct. 21, 2019. Dilution was completed as followed:

• In new PCR strip tubes, added 20 μl of previously made annealed adapter stocks

• Added 20 μl of Nuclease free water to annealed adapter stocks for 40 μl total of 20 μM adapter stocks

• Prepared adapter ligation master mix:
  • Ligation buffer: 15 μl * 9.5 = 142.5 μl
  • DNA ligase: 5 μl * 9.5 = 47.5 μl
  • Nuclease free water: 2.5 μl * 9.5 = 23.75 μl
• Added 22.5 μl of ligation master mix and appropriately planned adapters to each sample. Adapters were added last to minimize adapter-adapter ligation.

Sample	LMM	Adapter
1.2	22.5μl	2.5μl 2
1.6	22.5μl	2.5μl 6
2.6	22.5μl	2.5μl 4
3.9	22.5μl	2.5μl 5
4.4	22.5μl	2.5μl 10
4.7	22.5μl	2.5μl 1
5.6	22.5μl	2.5μl 10
5.8	22.5μl	2.5μl 12

• Pipetted up and down to mix - pipette set to 50 μl
  • spin down
• Incubated samples on shaker at room temp for 1 hour

0.8X Cleanup

• After incubation, added 44 μl of KAPA pure beads to each sample and pipetted up and down 10 times to mix (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 200 μl 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 200 μl 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 12.5 μl 10 mM Tris HCl pH 8 and incubated tubes on shaker for 5 minutes
• Placed tubes back onto the magnet plate and removed supernatant when clear to new labeled PCR strip tubes

Library Amplification

• Every 5 samples get a different index primer pair for amplification. For these 8 samples, 7 different master mixes were made:
• Amp MM A - Sample 1.2
  • 12.5 μl HotStart Ready mix
  • 1.25 μl 501 primer
  • 1.25 μl 701 primer
• Amp MM B - Sample 1.6
  • 12.5 μl HotStart Ready mix
  • 1.25 μl 502 primer
  • 1.25 μl 702 primer
• Amp MM C - Sample 2.6
  • 12.5 μl HotStart Ready mix
  • 1.25 μl 504 primer
  • 1.25 μl 704 primer
• Amp MM D - Sample 3.9
  • 12.5 μl HotStart Ready mix
  • 1.25 μl 506 primer
  • 1.25 μl 706 primer
• Amp MM E - Sample 4.4
  • 12.5 μl HotStart Ready mix
  • 1.25 μl 507 primer
  • 1.25 μl 707 primer
• Amp MM F - Sample 4.7
  • 12.5 μl HotStart Ready mix
  • 1.25 μl 508 primer
  • 1.25 μl 708 primer
• Amp MM G - Samples 5.6 & 5.8
  • 12.5 μl HotStart Ready mix * 2 = 25 μl
  • 1.25 μl 510 primer * 2 = 2.5 μl
  • 1.25 μl 710 primer * 2 = 2.5 μl
• Prepared new PCR tubes for the amplification with the following:

Sample	volume adapter added DNA of sample	volume of Amp MM
1.2	10μl	15μl Amp MM A
1.6	10μl	15μl Amp MM B
2.6	10μl	15μl Amp MM C
3.9	10μl	15μl Amp MM D
4.4	10μl	15μl Amp MM E
4.7	10μl	15μl Amp MM F
5.6	10μl	15μl Amp MM G
5.8	10μl	15μl Amp MM G

• Vortexed and spun down
• Placed samples in the thermocycler Genomic PCR program

1X Cleanup

• After PCR, added 25 μl of KAPA pure beads to each sample and pipetted up and down 10 times to mix (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 200 μl 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 200 μl 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 16 μl 10 mM Tris HCl pH 8 and incubated tubes on shaker for 5 minutes
• Placed tubes back onto the magnet plate and removed supernatant when clear to new labeled PCR strip tubes

Qubit and TapeStation

Completed on Oct. 16, 2019

• Followed Qubit protocol for BR DNA

Sample	Avg ng/μl
Std 1	191 RFU
Std 2	25292 RFU
1.2	68.2
1.6	106.5
2.6	97.4
3.9	100
4.4	91.2
4.7	101
5.6	46.8
5.8	66.4

• Followed tapestation protocol for D5000 tapes on 4 representative samples to check

See full report here

Sample 1.2:

Sample 3.9:

Sample 5.6:

Sample 5.8: