Category Archives: Notes

Fall 2016 Committee Meeting

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I had my Fall 2016 committee meeting on Thursday, Dec. 8. Pretty productive, with some good discussion about what to do going forward with my samples from this summer’s field season. Slides can be found here.

Goals December 2016

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  • Prepare for committee meeting on Dec. 8
    • Run EEMS, PCA, Structure, Bayescan, Migrate-N on cleaned, “final” denovo GBS assembly
    • Take stock of how many sites I have pH/temperature/salinity data for.
    • Create slides to discuss work from Summer 2016, planned transcriptomics
      • Graph survival in individual experiments
    • Create slides to discuss status of work from Summer 2015
  • Adult Olympia oyster experiment
    • Update spreadsheet with notes from final sampling day at Manchester
    • Have Roberts lab ship samples
  • Alpheus paper
    • Write discussion section
    • Make poster for SICB
  • 2bRAD Olympia oyster project
    • finish libraries of redos to send off for sequencing (by Dec. 20!)

Wet Lab posts on Benchling

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A few weeks ago I made 2 more Genotype-by-Sequencing libraries for my Olympia Oyster population structure study, made up mostly of redos and some samples from the sister species Ostrea conchaphila. I tried out the free version of Benchling for it and really liked the interface and how it organizes protocols with your notes. I’ll continue using it for my wet lab notes and post the links to my entries here. All field work posts and analysis updates will still be posted here. The link to my Benchling is:

https://benchling.com/ksil91/

This link is on my About page as well.

Monday 8/10/15 and Tuesday 8/11/15

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Monday 8/10/15

Larvae tanks

  • NF_Tank2_160 (224) -> 10,500 total: 500 for DNA, rest to NF_SetB
  • NF_Tank2_160 (100) -> swimmers only
  • HC_Tank2_160 (224) -> 9,187 total: 500 for DNA, 5,187 added to HC_SetB, rest to cultch set
  • HC_Tank2_160 (100) -> swimmers only
  • SS_Tank2_160 (224) -> 11,900 total, added to SS cultch
  • SS_Tank2_160 (100) -> swimmers only

Tile Set

  • Did live/dead counts for both A and B
  • Rinsed tiles (have been doing this everyday I’m in)
  • New totals
    • NF_SetB: 45,137
    • HC_SetB: 60,000

New larvae

  • no new larvae

Tuesday 8/11/15

Animal husbandry

  • Cleaned broodstock buckets
  • Rinsed cultch and tiles

Lab work

Extracted DNA from 24 larvae samples that would be good candidates for test 2b-RAD libraries. I chose sets of new larvae (“LC” for larvae catch), “160”s, and “224”‘s with 3-5 days in between.

Population Tank Family Size Date Storage Est. # Date extracted
1 Hood Canal LC >100 7/13/2015 75/95 EtOH 8/11/2015
2 Fidalgo Bay NA LC 100 7/13/2015 75% EtOH 8/11/2015
3 South Sound NA LC 100 7/13/2015 RNALater 8/11/2015
4 Hood Canal NA HC2 100 7/17/2015 RNALater 8/11/2015
5 Hood Canal NA LC 100 7/17/2015 RNALater 8/11/2015
6 South Sound NA LC 100 7/17/2015 RNALater 8/11/2015
7 Hood Canal NA LC 100 7/20/2015 RNALater 8/11/2015
8 Fidalgo Bay NA LC 100 7/20/2015 RNALater 8/11/2015
9 South Sound NA LC 100 7/20/2015 RNALater 8/11/2015
10 Hood Canal HC_Tank1_160 NA 160 7/20/2015 RNALater 8/11/2015
11 Fidalgo Bay NF_Tank1_new NA 160 7/20/2015 RNALater 8/11/2015
12 South Sound SS_Tank1_new NA 160 7/15/2015 RNALater 8/11/2015
13 Hood Canal HC_Tank1_new NA 160 7/24/2015 RNALater 8/11/2015
14 Fidalgo Bay NF_Tank1_new NA 160 7/24/2015 RNALater 8/11/2015
15 South Sound SS_Tank1_new NA 160 7/24/2015 RNALater 8/11/2015
16 Hood Canal HC_Tank1_new NA 160 7/27/2015 RNALater 8/11/2015
17 Fidalgo Bay NF_Tank1_new NA 160 7/27/2015 RNALater 8/11/2015
18 South Sound SS_Tank1_new NA 160 7/27/2015 RNALater 8/11/2015
19 Hood Canal HC_Tank2_160 224 8/3/2015 RNALater 8/11/2015
20 Fidalgo Bay NF_Tank2_160 224 8/3/2015 RNALater 8/11/2015
21 South Sound SS_Tank2_160 224 8/3/2015 RNALater 8/11/2015
22 Hood Canal HC_Tank2_160 >224 8/7/2015 RNALater 350 8/11/2015
23 Fidalgo Bay NF_Tank2_160 >224 8/7/2015 RNALater 504 8/11/2015
24 Oyster Bay SS_Tank2_160 >224 8/7/2015 RNALater 641 8/11/2015

I still have to go back over my notes to get estimated number for some of them. Storage was mostly in RNALater. A lot of the samples had some white precipitate on the bottom. If larvae weren’t  in the precipitate I sucked it out before adding the lysis buffer/proteinase K. Also had the same issue previously with larvae being buoyant in the RNALater even after a spindown. Halfway through trying to siphon off as much liquid as I could, I did some research and found that addition of some ice-cold PBS will change the density of the liquid and allow the larvae to settle out. This worked really well and was done for 2,10,13,15,19,20,21,24. It also dissolved most of the white precipitate.

Followed the protocol I listed here, with a 2.5 hour digest. Could not findd the gel rig set-up (found out later it’s in a different building).

Monday 8/3/15

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Setting System

Last week, I decided to make a 2nd tank with tiles for each of the populations. This stemmed from not seeing as many setters as I’d like on the tiles in the 1st set-up, and because there were still so many in the “160” tanks. Also, having 2 tanks per population can mitigate “tank effects”- possible tank-specific differences that might confound population differences. I picked up some more PVC sheet, had it cut into 4″ x 4″ tiles at the store, and roughed them up in between today’s tank cleanings. To ensure enough larvae set per tile, I’m going to add up to 60,000 larvae per tank over the course of 1 week. While this means some larvae will be a few more days older than others, after an extended period of time this will be insignificant for measuring growth rate.

Did counts for NF_SetA and HC_SetA to get an idea of how many were left to set (ran out of time for SS_SetA).

Larvae tanks

Measured out the “160” tanks over 224, 200, and 100 to get an idea of how many 224s I will get over the next week.

  • NF_Tank2_160 (224) -> 13,406 total: 500 for DNA, 12,906 to NF_SetB
  • NF_Tank2_160 (200)
  • NF_Tank2_160 (100) -> swimmers only added back
  • NF_Tank1_new (100) -> swimmers only added back
  • NF_Tank1_new (160) -> NF_Tank2_160
  • HC_Tank2_160 (224) -> 31,400 total: 500 for DNA, 30,900 added to HC_SetB
  • HC_Tank2_160 (200)
  • HC_Tank2_160 (100) -> swimmers only
  • HC_Tank1_new (100) -> swimmers only
  • HC_Tank1_new (160) -> HC_Tank2_160
  • SS_Tank2_160 (224) -> 20,200 total, 500 to DNA, 19,700 added to SS_SetB
  • SS_Tank2_160 (100) -> swimmers only
  • SS_Tank1_new (100) -> swimmers only
  • SS_Tank1_new (160) -> SS_Tank2_160

Friday 7/31/15

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Nine larvae/setting tanks to clean! Michael and I had an evening flight out east for his cousin’s wedding, so today was a pretty rushed day trying to get everything done and still make the 4:05p ferry.

Larvae tanks

  • NF_Tank2_160 (224) -> 31,200 total: 500 for DNA, 11,000 to cultch set B, 19,700 dumped out
  • NF_Tank2_160 (100) -> swimmers only added back
  • NF_Tank1_new (100) -> swimmers only added back
  • NF_Tank1_new (160) -> NF_Tank2_160
  • HC_Tank2_160 (224) -> 17,281 total: 500 for DNA, 11,000 added to cultch set B, 5,781 added back to HC_Tank2_160
  • HC_Tank2_160 (100) -> swimmers only
  • HC_Tank1_new (100) -> swimmers only
  • HC_Tank1_new (160) -> HC_Tank2_160
  • SS_Tank2_160 (224) -> 11,537 total, 537 to DNA, 11,000 to cultch set B
  • SS_Tank2_160 (100) -> swimmers only
  • SS_Tank1_new (100) -> swimmers only
  • SS_Tank1_new (160) -> SS_Tank2_160

Cultch Setting System

Set up a 2nd silo replicate for all 3 populations. As SS only had ~11,000 224-sized larvae, I added 11,000 larvae to each of the silos. 5,781 extra HC larvae were added back to HC_Tank2_160. With NF, there were ~9,000 dead 224-sized larvae- most likely due to my adding the the ones I wasn’t using back in to Tank2_160. If a larva that is ready to set can’t find an appropriate surface, it will eventually die. I dumped out the 19,700 larvae that were not used in the cultch set (see previous post for my regret about this).

New larvae

Only saw some larvae in HC and SS. I ran out of time to take samples for counting, so just screened out the larvae, cleaned the broodstock buckets, and dumped the larvae back into the buckets.

Friday 7-24-15 (S. Roberts)

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On Wednesday night I left to spend a few days in Chicago to start setting up the Committee on Evolutionary Biology’s new high performance computer, so Steven Roberts and Sam White came to the hatchery to do the Friday cleaning and counting. I wrote up some instructions– also good exercise so we can reference them when writing up Materials and Methods. Check out their great write-up here (lots of pictures!).

Sequencing Results from Nov. 22

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Crassostrea gigas:

  • BC3_6
  • BC3_5
  • BC3_8
  • all from Harris Point, Vancouver Island (of course they’d be from the site that cost $300 extra to go to)

Ostrea

  • BC2
    • 1
    • 2
    • 3
    • 4
    • 5
    • 6
    • BC2_7
    • 8
    • 13
    • 14
  • BC1
    • 1
    • 4
    • 2
    • 5
    • 7
    • 8
    • 9
    • 10
    • BC1_11
    • BC1_12
  • BC3
    • 1
    • 2
    • BC3_3
    • 4
    • 9
  • CA4
    • 1
    • 2
    • 3
    • 7
    • CA4_8
    • 9
    • 11
    • 12
    • 13
    • 14
  • OR2
    • 1
    • 2
    • OR2_13
    • 14

Sequences were too low of quality (b indicates reverse)

  • BC1_1b
  • BC2_13b
  • BC2_14b
  • BC2_1b
  • BC2_2b
  • BC2_8b
  • BC3_11a and b
  • BC3_7ab
  • CA4_11b
  • CA4_13b
  • CA4_14b

Realized I didn’t need to do forward and reverse because sequences are so different between Crassostrea and Ostrea that I do not need to confidently call every single nucleotide.