Hybridization of sequence capture probes and adult oyster DNA

Steps for completing the library preparation of adult oyster DNA can be accessed here:

• Part 1
• Part 2
• Part 3
• Part 4

Steps for completing the probe synthesis can be accessed here

Pool Adult DNA library samples into 5 capture pools

Completed on December 10, 2019

Samples were assigned to a specific capture pool prior to DNA library prep. Each sample in a capture pool has a unique index primer pair and adapter combo. This will allow us to differentiate the samples following sequencing.

• Pool 200 ng of each DNA library

Capture 1

Sample	Vol. of DNA for 200 ng (ul)
1.1	1.74
1.6	1.89
2.1	1.67
2.6	2.06
3.1	1.80
3.6	1.96
4.1	1.52
4.6	2.44
5.1	1.77
5.6	4.29
Total	21.14

Capture 2

Sample	Vol. of DNA for 200 ng (ul)
1.2	2.94
1.7	1.71
2.2	1.34
2.7	1.61
3.2	1.54
3.7	2.21
4.2	1.27
4.7	2.00
5.2	1.37
5.7	1.77
Total	17.76

Capture 3

Sample	Vol. of DNA for 200 ng (ul)
1.3	1.83
1.8	1.32
2.3	1.96
2.8	2.43
3.3	2.29
3.8	2.03
4.3	1.85
4.8	1.69
5.3	1.27
5.8	3.04
Total	19.71

Capture 4

Sample	Vol. of DNA for 200 ng (ul)
1.4	1.71
1.9	1.53
2.4	1.77
2.9	1.47
3.4	1.64
3.9	2.00
4.4	2.20
4.9	1.38
5.4	1.31
5.9	1.64
Total	16.65

Capture 5

Sample	Vol. of DNA for 200 ng (ul)
1.5	1.82
1.10	1.53
2.5	1.40
2.10	1.59
3.5	1.60
3.10	2.98
4.5	1.64
4.10	1.53
5.5	1.36
5.10	1.54
Total	16.99

Hybridization

Completed on December 10, 2019

• Add 500 ng of probes to each pooled capture
  • 500 ng/68.8 ng/ul = 7.27 ul
• Add nuclease-free water to bring volume up to 29.37

Sample	Vol. pooled w/ probes (ul)	Vol. of water (ul)
Capture 1	28.41	0.96
Capture 2	25.03	4.34
Capture 3	26.98	2.39
Capture 4	23.92	5.45
Capture 5	24.26	5.11

• Make Hybridization mastermix:
  • 15 ul of SSC (20X) x 5.3 samples = 79.5 ul
  • 0.5 ul of EDTA (500 mM) x 5.3 samples = 2.65 ul
  • 0.5 ul of SDS (10%) x 5.3 samples = 2.65 ul
  • 2.00 ul of Denhardt’s solution x 5.3 samples = 10.6 ul
  • 0.63 ul Cot-1 DNA (1 mg/ml) x 5.3 samples = 3.34 ul
  • 0.5 ul of Blocking oligo 1 (200 uM) x 5.3 samples = 2.65 ul
  • 0.5 ul of Blocking oligo 2 (200 uM) x 5.3 samples = 2.65 ul
  • 0.5 ul of Blocking oligo 3 (200 uM) x 5.3 samples = 2.65 ul
  • 0.5 ul of Blocking oligo 4 (200 uM) x 5.3 samples = 2.65 ul
• Add 20.63 ul of mastermix to each tube and pipette to mix
• Put samples in thermocycler using “Hybridization” program for 10 minutes
• Put tube rack with samples in plastic bag with damp paper towel and transfer to already warmed incubator genie
  • Set at 65 deg C rocking at 10 speed for 48 hours
  • Vortex and spin down samples after 24 hours and replace the paper towel with a fresh damp one

Wash and Capture

Completed on December 12, 2019

Creating various wash solutions for washes of the hybridized probes and adult DNA libraries

• Create TEN solution - need 5,500 ul
  • 0.5 ul of 500 mM EDTA x 5.5 samples = 2.75 ul x 4 uses = 11 ul
  • 50 ul 5 M NaCl x 5.5 samples = 275 ul x 4 uses = 1100 ul
  • 199.5 ul 10 mM Tris HCl pH 7.5 x 5.5 samples = 1097.25 ul x 4 uses = 4389 ul
• Solution 1 (1X SSC, 0.1% SDS) - need 2,200 ul
  • 10 ul of 20X SSC x 5.5 samples = 55 ul x 2 uses = 110 ul
  • 2 ul of 10% SDS x 5.5 samples = 11 ul x 2 uses = 22 ul
  • 188 ul of nuclease-free water x 5.5 samples = 1034 ul x 2 uses = 2068 ul
  • warmed to 65 deg C in thermomixer, put in 2 1.5 ml tubes with 1,100 ul in each
• Solution 2 (0.5X SSC, 0.1% SDS) - need 1,100 ul
  • 5 ul of 20X SSC x 5.5 samples = 27.5 ul
  • 2 ul of 10% SDS x 5.5 samples = 11 ul
  • 193 ul of nuclease-free water x 5.5 samples = 1061.5 ul
• Solution 3 (0.1X SSC, 0.1% SDS) - need 1,100 ul
  • 1 ul of 20X SSC x 5.5 samples = 5.5 ul
  • 2 ul of 10% SDS x 5.5 samples = 11 ul
  • 197 ul of nuclease-free water x 5.5 samples = 1083.5 ul

__Prepare Dynabeads__

• Resuspend Dynabeads M-280 beads first with p200
• Make 5 1.5-ml tubes with 10 ul of resuspended beads in each
• Add 250 ul of prepared TEN solution to each tube - pipette to mix
• Place tubes on magnet and remove supernatant when clear
• Remove the tubes from the magnet and resuspend beads in 250 ul of TEN solution
• Repeat the previous three wash steps two more times for 3 washes total
• Resuspend the beads in 250 ul of TEN solution

__Capture__

• After the 48 hour hybridization incubation, take the hybridized pools out of the incubator and spin down
• Add 50 ul of each of the 5 hybridization mixtures to each of 5 washed and prepared Dynabead PCR tubes (separate tube for each hybridization mixture)
• Pipette to mix and incubate on shaker at room temperature for 30 minutes
• Place Solution 1 in thermomixer to heat up to 65 deg C
• Make PCR strip tubes to save every wash - 25 total tubes
• After 30 minute incubation, place tubes on magnet
• Remove supernatant when clear and save in “Start” tube
• Resuspend beads in 200 ul of the 65 deg C heated Solution 1
• Place tubes in thermomixer set at 65 deg C for 15 minutes
• After 15 minute incubation, place tubes on magnet - beads had fallen to the bottom of the tube during the incubation
• Remove supernatant when clear and save in “Wash 1” tube
• Resuspend beads in 200 ul of the 65 deg C heated Solution 1
• Place tubes in thermomixer set at 65 deg C for 10 minutes
• After the 10 minute incubation, place tubes on magnet
• Remove supernatant when clear and save in “Wash 2” tube
• Resuspend beads in 200 ul of room temp Solution 2
• Place tubes in thermomixer set at 65 deg C for 10 minutes
• After the 10 minute incubation, place tubes on magnet
• Remove supernatant when clear and save in “Wash 3” tube
• Resuspend beads in 200 ul of room temp Solution 3
• Place tubes in thermomixer set at 65 deg C for 10 minutes
• Turn on thermomixer and turn on “80 deg C hold” program
• Place 200 ul of nuclease-free water in 80 deg C thermomixer
• After 10 minute incubation, place tubes on magnet
• Remove supernatant when clear and save in “Wash 4” tube
• Resuspend beads in 22 ul of 80 deg C heated nuclease-free water
• Place tubes in thermomixer set at 80 deg C for 10 minutes

__PCR amplification__

• Make PCR mastermix for amplification:
  • 12.5 ul of KAPA HiFi Hotstart Readymix x 5.2 samples = 65 ul
  • 2.5 ul of KAPA universal primer mix x 5.2 samples = 13 ul
• Aliquot 15 ul of PCR mastermix to each of 5 new PCR tubes labeled for each capture
• After the 10 minute incubation, spin down the Dynabead tubes and place on magnet
• Save supernatant when clear as captured DNA into new strip tubes
  • more than 22 ul in each tube, possibly leftover from the other washes?
  • the beads were resuspended in 20 ul of nuclease-free water and incubated for 10 minutes then saved (clear supernatant removed from beads) just to be safe
• Transfer 10 ul of the captured DNA to corresponding PCR amp tubes - vortex and spin down
• Place tubes in thermocycler for “post-capture 12-cycle PCR” program
• *Place the other tubes (all saved washes and remaining 10 ul of captured DNA) in -20 deg C freezer
  • Saved in box labeled EecSeq Samples etc.

1X Beadclean

• After PCR, perform 1X beadclean
• Made fresh 80% EtOH before starting
• Pulled KAPA Pure Beads out of fridge to warm to room temperature for ~30 minutes
• Added 25 ul of KAPA Pure Beads to sample and pipetted 10 times to mix
• Incubate at room temperature on shaker for 15 minutes
• Place tube on magnet, remove supernatant when clear
• Add 200 ul of 80% EtOH while sample is still on magnet
  • Remove clear supernatant
• Repeat previous step
  • Remove all of clear supernatant
• Resuspend the beads in 25 ul 10 mM Tris HCl pH 8 and incubate for 5 minutes
• Put on magnet and transfer all 25 ul of the clear supernatant to a new labeled tube

Qubit dsDNA High Sensitivity assay

The captured pools were quantified following Qubit protocol for BR DNA

• 199 ul x 7.5 samples = 1492.5 ul of Buffer
• 1 ul x 7.5 samples = 7.5 ul of reagent

Sample	Avg ng/μl
Std 1	46 RFU
Std 2	23673 RFU
Capture1	18.3
Capture2	18.6
Capture3	25
Capture4	18.1
Capture5	30.2

Captures were also run on the TapeStation following tapestation protocol for D5000 tapes.

See full report here.

These pools are ready to be sent off for sequencing!!

Sending samples off for sequencing

Completed on December 18, 2019

• Label tubes both caps and sides
  • NBAOcapture1
  • NBAOcapture2
  • NBAOcapture3
  • NBAOcapture4
  • NBAOcapture5
• Transfer 15 ul of the captured DNA pools to each of the labeled tubes