Presenting at Evolution and Updating Github

For the past 3 weeks I’ve been hanging around the Pacific Northwest, first going out to Tatoosh Island with my adviser, followed with some hiking, and then finishing up at the Evolution meeting in Portland, OR.  I gave an oral presentation of the first chapter of my dissertation on the population structure of Olympia oysters that you can check out here, and a poster of a paper that’s going to be submitted soon looking at adaptive differences among 3 populations of oysters in Puget Sound, WA (here). I’ve also been cleaning up my Github so that scripts and notebooks are better organized by project and have more documentation to them. In particular, I cleaned up the notebooks detailing the figures I made in my talk at Evolution.

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.

Submitted 2bRAD library for sequencing!

Yesterday I submitted a 2bRAD library for sequencing at the UChicago Genomics Core, with an anticipated turn-around time of 1-2 weeks. The details of the library prep can be found at this Benchling note. At the top of the note there is also a tab for the protocol I followed, with the specific master mixes made for this library (one of my favorite features of Benchling).

Goals December 2016

  • 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!)

Exploring GBS data and some file conversions

I’ve been working on organizing my scripts to analyze GBS data for my dissertation chapter on the phylogeography of the Olympia oyster. Still a lot of work to do, but here’s a couple of notebooks. All notebooks can be found at my Github under 2016_Notebooks and the scripts referenced in them can be found in my Github’s Scripts repository.

GBS_File_Conversions.ipynb: I’m planning on using this notebook to describe various file conversion scripts and code snippets I’ve written to work with. May end up splitting them into separate notebooks, but we’ll see. Currently, it describes how to add population information to the Structure (.str) output from ipyrad/pyrad, whether that be integers (required by the actual Structure program), or strings (useful for programs like Adegenet in R). It also describes how to take a .geno or .str file and split it up into many .geno files for looking at pairwise Fst and nucleotide diversity in R.

Exploring_GBS_Data_With_R.ipynb: This notebook has snippets of code used to explore pairwise Fst and nucleotide diversity (pi). Will eventually expand to include other R-based analysis methods.

 

Day 0 Sampling- Olympia Oyster OA Project

For my last morning in Washington state, Sam White and myself dissected 48 Olympia oysters for 3 different tissue types as part of an adult Olympia oyster/rock scallop ocean acidification experiment. Sam already wrote up a great summary of the day in his lab notebook. A list of all adult oysters sampled this summer, including those used in the adult OA experiment, can be found here or at the appropriate link under “Datasheets” in this lab notebook. This datasheet includes the date dissected, treatment tub, weight in grams, reproductive status at time of sampling, date of mortality (if oyster died prior to being dissected), and which tissues were samples. To measure size of oyster, I have labelled pictures of every oyster with a ruler in a Dropbox folder. I also wrote up a dissection protocol for subsequent sampling days.

Setting up for adult Olympia oyster OA experiment (20160907-11)

Wednesday 2016-09-07

In the morning, Natalie and I went to the Taylor Shellfish hatchery to pick up valves for the broodstock OA system. We got to Manchester around 12:30pm and worked on finishing up the plumbing and gluing the PVC. The hoses that were intended to be used to deliver water to the tubs didn’t fit easily on the valves, so we stuck one end in a toaster oven for a minute to make it easier to push on. We hooked up the manifolds to the OA system and let water run through overnight. Then we realized we hadn’t added in coupling for the algae injection port (D’oh!), so that needed to be addressed on Thursday.  We didn’t have time to give algae to the oysters or scallops, so both animals ended up without food overnight.

Oyster Mortalities

  • CA1T (1), BC1T (1), BC2T_058

Need to do: get brand name of valves

Thursday 2016-09-08

  • Used the rest of the white shellfish tags to label oysters that will be used in the adult OA experiment
    • Weighed oysters before adding tag and recorded their tray group during the larval experiment
    • Placed them in a single layer on the bottom of flow-through buckets with seawater and live algae overnight for putty to cure

20160909_132205

  • Checked for mortalities
    • 2 from OR1T (sampled 1)
    • 1 from OR2T (sampled)
  • Figured out the flow and food concentration with Ryan for the scallop tanks
    • Goal is to have at least 100,000 cells per mL of algae
    • Found that when valves are set to 50 the flow into each tub was ~0.85 L/min
    • Added couplings for the algae injection port on the flex hoses leading up to the manifolds
  • Starting around 5pm, took the scallops out of their fish totes and divided them up evenly amongst the 8 treatment tubs, with 11 or 12 scallops per tub. There were 4 fish totes corresponding to the 4 source populations.
    • Scuzz and some soft tissue epiphytes were gently wiped off with gloved hands. Scallops placed so they would not be touching each other
    • Temp of tubs: 15.12degC – 15.37degC, Salinity: 28.6
    • Tote A2 scallops spawned
      • Looked like eggs from 380, 311, 48, 381
    • Tote B2 spawned
      • just eggs from 312
Tub 1A Tub 1B
  • Tote 5: 305, 302
  • Tote 4: 363, 360, 244
  • Tote 3: 63, 45, 241, 57
  • Tote 1: 375, 391, 390
  • Tote 5: 310, 303
  • Tote 4: 240, 361, 358
  • Tote 3: 66, 36, 65
  • Tote 1: 373, 372, 379, 383
Tub 2A Tub 2B
  • Tote 5: 311
  • Tote 4: 246, 359, 237
  • Tote 3: 48, 53, 58, 64, 67
  • Tote 1: 392, 381, 380
  • Tote 5: 312
  • Tote 4: 236, 242, 248
  • Tote 3: 51, 53, 39, 56
  • Tote 1: 374, 370, 387
Tub 3A Tub 3B
  • Tote 5: 309, 300
  • Tote 4: 362, 234, 245
  • Tote 3: 44, 38, 43
  • Tote 1: 377, 388, 393, 384
  • Tote 5: 308, 301
  • Tote 4: 364, 233, 231
  • Tote 3: 37, 50, 54
  • Tote 1: 389, 386, 376
Tub 4A Tub 4B
  • Tote 5: 306, 304
  • Tote 4: 237, 239
  • Tote 3: 59, 52, 60
  • Tote 1: 371, 395, 385, 46
  • Tote 5: 307
  • Tote 4: 243, 247, 238
  • Tote 3: 41, 61, 62, 47
  • Tote 1: 382, 378, 394

 

Friday 2016-09-09

  • Checked for mortalities
    • BC 049
  • Divided up oysters and placed them into clam bags to hang in treatment tubs with scallops.
    • 12 British Columbia oysters per treatment tub
    • 10 California and 10 Oregon oysters per treatment tub
  • Dissected adductor muscle from extra BC oysters not used in OA experiment
    • Recorded weight, reproductive status, tray group, and took pics for size
    • BC1T A, B, 93, C, D, 101
    • BC2T A, 111, 110, B, C, D, E, 108
  • Checked algae in one A tub and 1 B tub using Coulter counter
    • 92,921 cells/ml, 99,070 cells/ml

 

Sunday 2016-09-11

  • Recorded which oysters were in which experiment tub and made sure numbers were balanced, in case an oyster gets misplaced when cleaning
  • Checked for mortalities
    • 024W (CA)
    • 058W
  • Weighed and took pictures for size of any oysters I did not already have data for
  • Dissected adductor muscle into RNALater from 12 oysters that were in tray OR1T during larval experiment
    • Labelled OR1T A-L
  • Processed the rest of the tiles from the acidification larval experiment
    • These are 4.5in diameter PVC tiles that were roughed up on both sides with sandpaper and placed with oyster larvae 14 days post-release
      • Only added if at least 20% survival in treatment
    • To process them I counted the number of live and dead oysters per tile, took a pic of both sides with a ruler, and scraped off juveniles into RNALater if there were at least 5 living oysters on the tile