Sam’s Notebook: Genome Annotation – Olympia oyster genome complete – brief note

0000-0002-2747-368X

Whoa! Genome annotation using Jetstream/WQ-MAKER that I started this morning is complete!! Only 7hrs!

More detailed entry coming once I move files off of Jetstream and have a chance to look at things.

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Yaamini’s Notebook: Gigas Broodstock DNA Extraction Part 3

Protocol test: Round 2

Here we go again!

Step 0: Prepare for extractions

  • Preheat the thermomixer to 56ºC
  • Clean scalpel in 10% bleach solution

Step 1: Cut a tissue sample from the parrafin block

  • I used Graham’s scale, which was way more sensitive
  • Tared the scale with a piece of weigh paper
  • Used a scooping thing to cut and scoop out parrafin-embedded tissue
    • Today I carved out waaay less tissue than I did yesterday! I think I definitely had too much tissue yesterday, which could have caused the lower yield
  • Set the tissue on the weigh paper
  • Weighed 0.0201 g of tissue

Step 2: Cut the block into smaller pieces and place them into a 2 mL round-bottomed processing tube

  • Following the protocol’s suggestions, I cut the block into much smaller pieces than I did yesterday

Step 3: Add 1 mL xylene to the sample. Vortex vigorously for 20 s, and incubate for 3 min on the benchtop

Step 4: Centrifuge at maximum speed for 3 minutes (but do not exceed 20,000 x g)

  • Maximum speed on the centrifuge was 13,000 RPM x g

Step 5: Remove the supernatant by pipetting. Do not remove any of the pellet

Step 6: Add 1 mL of ethanol (96%-100%, purity grade p.a.) to the pellet, and mix by vortexing for 20 s.

Step 7-8: Repeat Steps 4-5

Step 9: Open the tube and incubate at room temperature for 10 minutes, or until all residual alcohol has evaporated

  • I incubated the sample for 25 minutes, just like yesterday

Step 10: Resuspend the pellet in 180 µL Buffer TD1.

Step 11: Add one stainless steel bead to each 2 mL processing tube, and place the tubes in the TissueLyser Adapter Set 2 x 24.

  • Added about 50 µL of glass beads (1 small spatula full) into the 2 mL sample tube

Step 12: Operate the TissueLyser for 20 s at 15 Hz

  • Instead, I vortexed the sample and glass beads at maximum speed for 25 s

Step 13: Carefully pipet lysates into new 1.5 mL microcentrifuge-safelock tubes

  • The protocol suggested that I vortex the lysates vigorously to improve yield, so I vortexed the lysates for 20 s

Step 14: Add 20 µL proteinkinase K, and mix by pulse-vortexing for 15s

Step 15: Incubate for 1 hour at 56ºC using a shaker-incubater at 1400 rpm.

  • I used the Genome Sciences thermomixer, set to 56ºC and 1400 rpm, for one hour.
  • After the incubation ended, I increased the temperature to 80ºC to prepare for Step 18

Step 16: Briefly centrifuge the microcentrifuge-safelock tube to remove drops from the inside of the lid

  • I centrifuged the tube for 15 s at 13,000 RPM x g
  • There was no gelatinous pellet after incubation and centrifuging, so Proteinkinase K digestion did not go horribly

Step 17: Add 4 µL RNase A (100 mg/mL), mix by vortexing, and incubate for 2 min at room temperature

  • I added RNase A
  • Vortexed for 20 s at maximum speed
  • Incubated at room temperature for 2 minutes
  • Incubated at room temperature an extra 4.25 min while I was waiting for the thermomixer to reach 80ºC

Step 18: Incubate for 60 min at 80ºC at 1400 rpm

Step 19: Repeat Step 16

Step 20: Add 200 µL Buffer TD2, and mix by pulse-vortexing for 15 s

Step 21: Add 200 µL ethanol (96-100%) to the sample, and mix thoroughly by vortexing

  • I mixed for 35 s using a vortex at maximum speed

Step 22: Repeat Step 16

Step 23: Pipet the sample, including any precipitate that may have formed, into the PAXgene DNA spin column placed in a 2 mL processing tube, and centrifuge for 1 min at 6000 x g. Plae the spin column in a new 2 mL procesing tube, and discard the old processing tube containing flow-through.

Step 24: Pipet 500 µL Buffer TD3 into the PAXgene DNA spin column, and centrifuge for 1 min at 6,000 x g. Place the spin column in a new 2 mL processing tube, and discard the old processing tube containing flow-through.

Step 25: Pipet 500 µL Buffer TD4 into the PAXgene DNA spin column, and centrifuge for 1 min at 6,000 x g. Place the spin column in a new 2 mL processing tube, and discard the old processing tube containing flow-through.

Step 26: Centrifuge for 3 min at maximum speed (but do not exceed 20,000 x g) to dry the membrane completely.

Step 27: Discard the processing tube containing flow-through. Place PAXgene DNA spin column in a 1.5 mL microcentrifuge tube, and pipet 50-200 µL Buffer TD5 directly onto the PAXgene DNA spin column membrane. Centrifuge for 1 min at maximum speed (but do not exceed 20,000 x g) to elute the DNA.

  • I pipetted 50 µL on the spin column membrane and let it incubate for 5 minutes, then centrifuged for one minute at 13,000 RPM x g

Step 28: Assess DNA yield with the Qubit

  • Obtain dsDNA BR standards from fridge
  • Prepare the master solution, using a 1:200 ratio of dsDNA BR buffer to dye. The master solution is used for the two standards and the samples
    • Each sample needs 200 µL of master solution. Since I only had three tubes (2 standards, 1 sample), I needed 600 µL of solution
    • I prepared 660 µL of solution, using 656.7 µL buffer and 3.3 µL dye. 660 µL solution * 0.5 / 100 = 3.3 µL dye
  • Pipet 200 µL master solution into each Qubit assay tube
  • Add 10 µL of the correct standard to the standard assay tube
  • Add 5 µL of sample to the sample tube
  • Vortex the tubes for 2-3 seconds
  • Incubate tubes at room temperature for 2 minutes
  • Use Quibit to quantify yield
    • It’s pretty easy to follow the instructions on the machine! You select your assay (dsDNA BR), read standards, then load in the samples

Results

Somehow I ended up with a lower yield than last time…1.16 ng/µL. I need 6000 ng total per sample to do MBD-Seq, which means 130 ng/µL. I need 100x more DNA!

Going forward

I posted another comment in this issue asking for guidance. One possible solution could be to increase the time I vortex with glass beads. I’ll try Thursday?

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Sam’s Notebook: Genome Annotation – Olympia oyster genome using WQ-MAKER Instance on Jetstream

0000-0002-2747-368X

Yesterday, our Xsede Startup Application (Google Doc) got approval for 100,000 Service Units (SUs) and 1TB of disk space on Xsede/Atmosphere/Jetstream (or, whatever it’s actually called!). The approval happened within an hour of submitting the application!

Here’s a copy of the approval notice:

Dear Dr. Roberts:

Your recently submitted an XSEDE Startup request has been reviewed and approved.

PI: Steven Roberts, University of Washington

Request: Annotation of Olympia oyster (Ostrea lurida) and Pacific geoduck (Panopea generosa) genomes using WQ_MAKER on Jetstream cloud.

Request Number: MCB180124 (New)

Start Date: N/A

End Date: 2019-08-05

Awarded Resources: IU/TACC (Jetstream): 100,000.0 SUs

IU/TACC Storage (Jetstream Storage): 1,000.0 GB

Allocations Admin Comments:

The estimated value of these awarded resources is $14,890.00. The allocation of these resources represents a considerable investment by the NSF in advanced computing infrastructure for U.S. The dollar value of your allocation is estimated from the NSF awards supporting the allocated resources.

If XSEDE Extended Collaborative Support (ECSS) assistance was recommended by the review panel, you will be contacted by the ECSS team within the next two weeks to begin discussing this collaboration.

For details about the decision and reviewer comments, please see below or go to the XSEDE User Portal (https://ift.tt/2M6Cm56), login, click on the ALLOCATIONS tab, then click on Submit/Review Request. Once there you will see your recently awarded research request listed on the right under the section ‘Approved’. Please select the view action to see reviewer comments along with the notes from the review meeting and any additional comments from the Allocations administrator.

By default the PI and all co-PIs will be added to the resources awarded. If this is an award on a renewal request, current users will have their account end dates modified to reflect the new end date of this award. PIs, co-PIs, or Allocation Managers can add users to or remove users from resources on this project by logging into the portal (https://ift.tt/2M6Cm56) and using the ‘Add/Remove User’ form.

Share the impact of XSEDE! In exchange for access to the XSEDE ecosystem, we ask that all users let us know what XSEDE has helped you achieve:

  • For all publications, please acknowledge use of XSEDE and allocated resources by citing the XSEDE paper (https://ift.tt/2OkPwZD) and also add your publications to your user profile.
  • Tell us about your achievements (https://ift.tt/2vseOxV).
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For question regarding this decision, please contact help@xsede.org.

Best regards,
XSEDE Resource Allocations Service

Yaamini’s Notebook: Gigas Broodstock DNA Extraction Part 2

Testing the extraction protocol

Now that I had both xylene and a thermomixer, I could test the PAXgene Tissue DNA Kit.

Step 0: Prepare for extractions

  • Add ethanol to Buffer TD3 and TD4. The ethanol amount is specified on the container
  • Preheat the thermomixer to 56ºC
  • Label tubes for extractions
  • Clean scalpel in 10% bleach solution
  • Stuck the spin columns in the 4ºC fridge. They were supposed to be stored in the fridge upon arrival, but that didn’t happen :/

Step 1: Cut a tissue sample from the parrafin block

  • Tared a scale with a piece of weigh paper
  • Used a scooping thing to cut and scoop out parrafin-embedded tissue
  • Set the tissue on the weigh paper until I had 0.02 g of tissue
    • The weigh paper kept oscillating between 0.00, 0.01, and 0.02! I carved out more tissue than I did the first time so I’m confident I have enough tissue. I need to find a more sensitive scale to use so my breathing doesn’t affect the scale.

Step 2: Cut the block into smaller pieces and place them into a 2 mL round-bottomed processing tube

  • The block was already cut into smaller pieces, so I carefully folded the weigh paper and scooped the tissue into a 2 mL screwtop tube

Step 3: Add 1 mL xylene to the sample. Vortex vigorously for 20 s, and incubate for 3 min on the benchtop

Step 4: Centrifuge at maximum speed for 3 minutes (but do not exceed 20,000 x g)

  • Maximum speed on the centrifuge was 13,000 RPM x g

Step 5: Remove the supernatant by pipetting. Do not remove any of the pellet

Step 6: Add 1 mL of ethanol (96%-100%, purity grade p.a.) to the pellet, and mix by vortexing for 20 s.

Step 7-8: Repeat Steps 4-5

Step 9: Open the tube and incubate at room temperature for 10 minutes, or until all residual alcohol has evaporated

  • I incubated the samples for 25 minutes. I could incubate the samples at 37ºC for a shorter incubation time

Step 10: Resuspend the pellet in 180 µL Buffer TD1.

Step 11: Add one stainless steel bead to each 2 mL processing tube, and place the tubes in the TissueLyser Adapter Set 2 x 24.

  • Instead, I added about 50 µL of glass beads (1 small spatula full) into the 2 mL sample tube

Step 12: Operate the TissueLyser for 20 s at 15 Hz

  • Instead, I vortexed the sample and glass beads at maximum speed for 20 s

Step 13: Carefully pipet lysates into new 1.5 mL microcentrifuge-safelock tubes

Step 14: Add 20 µL proteinkinase K, and mix by pulse-vortexing for 15s

Step 15: Incubate for 1 hour at 56ºC using a shaker-incubater at 1400 rpm.

  • I used the Genome Sciences thermomixer, set to 56ºC and 1400 rpm, for one hour.
  • After the incubation ended, I increased the temperature to 80ºC to prepare for Step 18

Step 16: Briefly centrifuge the microcentrifuge-safelock tube to remove drops from the inside of the lid

  • I centrifuged the tube for 0.5 minutes at 13,000 RPM x g

Step 17: Add 4 µL RNase A (100 mg/mL), mix by vortexing, and incubate for 2 min at room temperature

  • This is an optional step to obtain RNA-free genomic DNA. Sam said it wouldn’t hurt anything if I did it!
  • I added RNase A (found in the chemical cabinet) to my sample
  • Vortexed for 20 s at maximum speed
  • Incubated at room temperature for 2 minutes
  • Incubated at room temperature an extra 5 minutes while I was waiting for the thermomixer to reach 80ºC

Step 18: Incubate for 60 min at 80ºC at 1400 rpm

Step 19: Repeat Step 16

Step 20: Add 200 µL Buffer TD2, and mix by pulse-vortexing for 15 s

Step 21: Add 200 µL ethanol (96-100%) to the sample, and mix thoroughly by vortexing

Step 22: Repeat Step 16

  • Some white precipitate formed and was present on the bottom of the tube

Step 23: Pipet the sample, including any precipitate that may have formed, into the PAXgene DNA spin column placed in a 2 mL processing tube, and centrifuge for 1 min at 6000 x g. Plae the spin column in a new 2 mL procesing tube, and discard the old processing tube containing flow-through.

Step 24: Pipet 500 µL Buffer TD3 into the PAXgene DNA spin column, and centrifuge for 1 min at 6,000 x g. Place the spin column in a new 2 mL processing tube, and discard the old processing tube containing flow-through.

Step 25: Pipet 500 µL Buffer TD4 into the PAXgene DNA spin column, and centrifuge for 1 min at 6,000 x g. Place the spin column in a new 2 mL processing tube, and discard the old processing tube containing flow-through.

Step 26: Centrifuge for 3 min at maximum speed (but do not exceed 20,000 x g) to dry the membrane completely.

Step 27: Discard the processing tube containing flow-through. Place PAXgene DNA spin column in a 1.5 mL microcentrifuge tube, and pipet 50-200 µL Buffer TD5 directly onto the PAXgene DNA spin column membrane. Centrifuge for 1 min at maximum speed (but do not exceed 20,000 x g) to elute the DNA.

  • I talked to Sam, and he suggested I add only 50 µL of Buffer TD5. While adding the maximum amount of elution buffer would increase my yield, adding 50 µL should still give me a 90% DNA yield with a higher concentration
  • Incubating the PAXgene DNA spin column loaded with Buffer TD5 for 5 min at room temperature before centrifugation generally increases DNA yield (so that’s what I did)

Step 28: Assess DNA yield with the Qubit

  • Obtain dsDNA BR standards from fridge
  • Prepare the master solution, using a 1:200 ratio of dsDNA BR buffer to dye. The master solution is used for the two standards and the samples
    • Each sample needs 200 µL of master solution. Since I only had three tubes (2 standards, 1 sample), I needed 600 µL of solution
    • I prepared 660 µL of solution, using 656.7 µL buffer and 3.3 µL dye. 660 µL solution * 0.5 / 100 = 3.3 µL dye
  • Pipet 200 µL master solution into each Qubit assay tube
  • Add 10 µL of the correct standard to the standard assay tube
  • Add 5 µL of sample to the sample tube
  • Vortex the tubes for 2-3 seconds
  • Incubate tubes at room temperature for 2 minutes
  • Use Quibit to quantify yield
    • It’s pretty easy to follow the instructions on the machine! You select your assay (dsDNA BR), read standards, then load in the samples

Results

I had a relatively low DNA yield of 3.05 ng/µL 😦 But at least I know the protocol works?

Going forward

  • While writing this lab notebook post, I realized I tossed the spin columns in the freezer instead of the fridge. So I ran upstairs, moved the spin columns to the fridge, and ran back down. Hopefully I didn’t ruin the spin columns.
  • I’m going to try this protocol again tomorrow with one more sample. I think I really need a better scale to properly measure the 0.02 g from each tissue
  • The protocol also has some suggestions:
    • Insufficient mixing of sample with Buffer TD2 and ethanol before binding: Mix sample first with Buffer TD2 and then with ethanol by pulse vortexing for 15 s each time before applying the sample to the PAXgene DNA spin column (Steps 20 and 21)
    • Insufficient lysis: Cut tissue into smaller piees to facilitate lysis (they were already pretty small…so I guess I’ll try again!). After lysis, vortex sample vigorously; this will not damage or reduce the size of the DNA. If a substantial gelatinous pellet remains after incubation and vortexing (not my case), extend incubation time at 56ºC for proteinase K digestion.

Hopefully tomorrow’s yields are higher!

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