Submitted 2bRAD Library 6 for Sequencing

I submitted another 2bRAD library today. This one has 25 samples of pooled Ostrea lurida larvae (and 3 samples from juvenile spat). Some of the samples correspond to the growth experiments done in Summer 2015, some of them correspond to the G2 South Sound oysters being used in Laura’s experiment, and some are just extras. The main goals of this library are to:

  1. See what pooled 2bRAD data “looks” like
  2. determine the amount of sequencing depth needed for confident genotyping (i.e. how many samples can go on a lane)
  3. determine if parentage can be assigned using 2bRAD sequencing from adults
    1. Bonus if successful- get an idea of how many parents contributed to my experiments

Details and notes from the library prep (including protocol) can be found at this Benchling note.

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Monday 8/3/15

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

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.

Thursday 7/30/15 (Lab work!)

I got to do some bona fide lab work today, which was a nice change of pace. I’ve been taking samples of larvae for DNA sequencing at various points throughout the experiment:

  • From all newly released larvae (either from each family or combined, depending on how I filtered them out)
  • From larvae in the “New” tanks that reach 160 microns
  • From larvae in the “160” tanks that reach 224 in size
  • Occasionally pooled larvae from a tank

These samples have mostly been stored at -20degC in .5-1 mL of RNALater, but duplicates of many were also stored in ethanol (1st in 75%, then in 95%). Earlier in the summer I wanted to do a test extraction to see if there was a particular storage method that worked best and figure out which extraction kit to use, but then the oysters needed maintenance 6 days a week and all of a sudden in was almost August. With the growth rate experiments and larval production essentially done, I finally had a day to do the test extraction.

Continue reading

Tuesday 7/28/15 and Wednesday 7/29/15

Tuesday 7/28/15

New Larvae

  • HC: 0
  • SS: some
  • NF: 0

Settlement

Set up an “overflow” setting system for larvae that do not go into the tile set-up. Silos (15 cm diameter, 20 cm height) with 180 micron screens are suspended in a tote with a draining outlet. 224 sized larvae are added into the silos along with 1-3 tablespoons of 450 micron cultch. An airstone is in the totes and water/algae is dripped into the silos. The water level is 13 cm deep, making the volume of water in the silo to be 2,296 cm^3 (equalling to 2,296 mL). I’ve been told that ~5 larvae/mL is good for this type of set-up, with no more than 10 larvae/mL. At 7 larvae/mL, the max to add to each silo would be 16,000 larvae.

Wednesday 7/29/15

Larvae tanks

  • NF_Tank2_160 (224) -> 56,700 total: 600 for DNA, 15,300 to cultch set, 41,400 to NF_Tank2_160
  • NF_Tank2_160 (100) -> swimmers only added back
  • NF_Tank1_new (100) 0> swimmers only added back
  • NF_Tank1_new (160) -> NF_Tank2_160
  • HC_Tank2_160 (224) -> 15,937 total: 600 for DNA, 15,300 added to cultch set
  • HC_Tank2_160 (100) -> swimmers only
  • HC_Tank1_new (100) -> swimmers only
  • HC_Tank1_new (160) -> HC_Tank2_160
  • SS_Tank2_160 (224) -> 9,675 total, 600 to DNA, 9,075 to cultch set
  • SS_Tank2_160 (100) -> swimmers only
  • SS_Tank1_new (100) -> swimmers only (a lot on bottom)
  • SS_Tank1_new (160) -> SS_Tank2_160

New larvae

  • Some new larvae from all 3 populations

Setting Systems

To clean the tile setting systems, I first fill up a clean, empty 100 L larval tank with seawater and fill up a misting sprayer designated for setters with fresh water. I empty a tile setting tank over a 100 micron screen to catch the larvae that haven’t set yet. I put these in a tripour beaker with seawater and place the tiles in the 100 L tank full of water. I clean the tank with Vortex and fill it up as quickly as possible. Meanwhile, I spray the tiles and poultry wire gently with freshwater before returning the tiles and larvae into the cleaned tank. This took a little getting used to, especially figuring out how to minimize the time the larvae sat in the beaker as they would try to set on the bottom if they were in there too long.

I initially thought that the cultch set would be an experiment to see if the populations have differential success in the number of single oysters produced. Because of this, I cared a lot about having replicate silos, adding similar numbers of larvae to each, adding larvae on the same day, adding the same amount of cultch to each, and randomizing the order of silos. This led to me not using all of the 224s that I screened out, particularly from NF which somehow peaked in the number of 224s before the other 2 groups. I’ve since realized that to truly make it a viable experiment would take a lot of work (especially in the number of silos needed once I started screening them out by size) and that the main benefit of the cultch set is to grow up F2 oysters for future experiments. So I regret throwing out some of the 224s, but should still have enough for the project goals.

  • NF: 15,300 added to Cultch_SetA
  • SS: 9,075 added to Cultch_SetA
  • HC: 15,300 added to Cultch_SetB

Tuesday 7/14/15 – Sunday 7/19/15

Tuesday 7-14-15

Today I primarily checked for new larvae and did a mortality count for the HC tanks (as these had some of the most culling of non-swimmers during the tank cleanings on Monday). I also drilled holes into my 10cmx10cm PVC tiles so that I can tie them on to a plastic coated wire mesh for the settlement stage.

Checking for larvae

  • Some: SS3,HC3,HC1,NF1,SS1,SS2,HC2(?)
  • Lots: NF3

Larval tank counts

  • HC_Tank2_160 (100)
  • HC_Tank1_new (100)
  • HC_Tank1_new (160) -> HC_Tank2_160

Wednesday 7-15-15

S. Roberts came in today to help out with cleaning, counting, and setting up the settlement system. He roughed up one side of the PVC tiles with sandpaper, so as to make that side more preferential to oyster settlement.

Larval tank counts

  • NF_Tank2_160
  • NF_Tank1_new (100)
  • NF_Tank1_new (160) -> NF_Tank2_160
  • HC_Tank2_160
  • HC_Tank1_new
  • HC_Tank1_new (160) -> HC_Tank2_160
  • SS_Tank2_160
  • SS_Tank1_new
  • SS_Tank1_new (160) -> SS_Tank2_160

New larvae

With the extra help today, I decided to do individual counts for each family. There were very few, with none for NF, making me think that the broodstock are winding down.

  • Some: SS3,HC2,HC3,HC4

Growth experiments

  • I took some more samples from the growth rate experiments today for size measurement, and will be taking down the 1st growth experiment on Friday.

Friday 7-17-15

The seawater was shut off for a couple hours today as they were doing some reorganization of the hatchery. I was able to clean and take samples for counting from the larvae tanks before the shut off and then finish the broodstock buckets afterwards. I made sure everyone had enough food during the break by splashing some extra algae in.

Larval tank counts

  • NF_Tank2_160
  • NF_Tank1_new (100)
  • NF_Tank1_new (160) -> NF_Tank2_160
  • HC_Tank2_160
  • HC_Tank1_new
  • HC_Tank1_new (160) -> HC_Tank2_160
  • SS_Tank2_160
  • SS_Tank1_new
  • SS_Tank1_new (160) -> SS_Tank2_160

New larvae

Well I was wrong by thinking that they were all winding down. There was 441,100 larvae from SS and 205,133 from HC, but barely any from NF.

  • Some: SS4,NF1,HC1,HC4
  • Lots: SS3,HC2,HC3,HC5,SS2

Salinity

Took samples of ~100 larvae from each of the 45 silos in the salinity experiment to do live/dead counts and save for taking pictures. Unfortunately Ryann, the PSRF intern helping me on the salinity project, and I ran out of time so I was just able to do a rough activity estimate (% swimming). I fixed the wells in 10% formalin and left covered in parafilm to look at next week.

Sunday 7/19/15

Came in for a couple hours on my way back from a camping trip in Olympic National Park. Cathy Pfister, my adviser from UChicago, stopped by for a tour of my setup and to catch up on how things are going. With Michael’s help, I also did the water changes for the 2nd growth experiment and the salinity experiment and changed out banjo filters.