Ronit’s Notebook: Adjusted qPCR Data, COX1 Relative Mitochondrial Abundance Plot

To account for N/A Cq values, I substituted in a Cq value of 45 wherever the Cq value was nonexistent. This allows for some initial analysis of the data to see which genes might warrant further work. Below are the adjusted qPCR plots. I also generated the COX1 Cq plot (not normalized to actin) to examine relative mitochondrial abundance between stressed and non-stressed diploids and triploids.


Shelly’s Notebook: Wed. Jan 30, 2019

Geoduck Broodstock Experiment

Gonad slices sent to HCS, Inc. for histology

List of samples sent:

Order form:

Shipped through FedEx as hazardous goods of expected quantities, each of the 5 cassette jars contained 30 mL of 70% Ethanol (Stabilizer from PAXgene kit).

Low pH conditioning plans

Steven and I discussed stopping the treatment on Friday and combining the low pH group crates (Tank 1 and 2) into one ambient tote and Tank 3 and 4 crates into one ambient tote. This will make room for more broodstock and will hopefully rescue the low pH conditioned animals so we can get them to spawn/strip spawn. See slack discussion.

Oyster Seed Proteomics

Map the proteins to 2019 Uniprot accessions

For re-mapping the fasta to 2019 Uniprot accessions like Steven did in the past, I started the jupyter notebook linked below on Ostrich and it’s currently still running:

Github desktop on Ostrich

Also got github desktop to work on Ostrich by downloading this older version that I found here. With the newest version, the graphics don’t work when you remote desktop in. But this older version works fine, probably because Ostrich is still running El Cap OS.

from shellytrigg

Shelly’s Notebook: Tues. Jan 30, 2019

Concentrating Geoduck Broodstock Hemocyte DNA for WGBS

Because the minimum concentration for WGBS with Genewiz is 20ng/ul, I pooled and precipitated the DNA samples from yesterday as follows:

  • I combined tank 3 samples 15 and 16, total volume ~200uL
  • I combined tank 2 samples 025 and 026, total volume ~200uL
  • I added 140uL isopropanol and 20uL 3M Sodium Acetate pH 5.2 to each ~200uL pooled sample
  • I vortexed to mix
  • I spun at 12000 rpm for 30 min at 4C
  • I removed the supernatent and added 500uL of 70% ethanol to each
  • I spun tubes for 10 min at 12000 rpm at 4C
  • I removed the supernatent and resuspended in 20uL EB (elution buffer from the EZNA kit, which I think is 10mM Tris, pH 8.5)

Qubit concentrations: Standard 2 = 100ng/ul

Animal ID Tank Treatment Conc. (ng/ul) Total DNA (ng)
15_16 3 amb 284 1230
025_026 2 low (pH 6.8) 53 830

We are going to send all of Tank 2 ‘025_026’ DNA and half of Tank 3 ‘15_16’ DNA, which Sam now has and planning to ship on Monday. The remaining Tank 3 ‘15_16’ DNA is in my box labeled ‘Shelly Trigg start date 10/11/2018’ in the -20C in rm 213.

Oyster seed proteomics time x temperature

Creating differential network visualizations

To create differential network visualizations, we need:

  1. protein relationships
    • this could be GO annotations, KEGG pathways, or even protein interaction information (although I’m not sure how conserved this is between species)
  2. a metric to quantify abundance changes
    • nodes could be colored by fold change and/or effect size to show up or down
    • node size could be relative to p-value, or even PCA loadings values
Calculating log fold change and p-values

Emma suggested calculating the ratio of ‘NUMSPECSTOT’ of each protein to the sum of NUMSPECSTOT for the sample. Then calculating log fold change. And using a proportions test for calculating the p-value.

I think all fold changes should be relative to day zero so that we can visualize what protein networks are active at each stage of development and compare differences between the two temperatures (sort of like what these guys did). For a figure, what I’m picturing is a 2 x 6 grid for the two different temperatures and 6 different days. And a network in each box of the grid showing proteins colored differently based on their fold change and sizes based on p-value significance.

Here is the R markdown file I made for calculating these. I used a Chi square proportions test for the p-values. So far I have only done one comparison (23C_day 3 to day 0).

Need to:

  • make a loop to do the rest of the comparisons
  • map the proteins to Uniprot accessions using an updated uniprot DB.
  • figure out what proteins are connected either by GO or physical interaction information

from shellytrigg

Laura’s Notebook: Oly OA gonad histology & spawning data options

I’ve come full circle with my data from the Olympia oyster adult OA exposure project from 2017. After agonizing over the data for months (…years?), I’m moving forward with population-specific analysis, focusing on the effect of low pH on gonad & fecundity, with some minor findings regarding the offspring. Until a couple days ago I was all set to just use data from the 6C pre-treatment temperature groups (overwintered at 6C), but since we are now looking at population specific effects I may want to include the 10C groups (overwintered at 10C). The reasons to include both temperature groups are a) very simliar gonad results in both groups, b) more spawning data which support the population-specific reproduction theme, and c) I can refer to this paper when I write my QuantSeq paper (since RNA from larvae was from 10-low pH and 6-amb pH). If I go this route I will not analyze the 10C survival/growth data.

I’ve prepared some new figure options, particularly for the gonad histology data:

6C gonad data only, showing dominant stage, sex, and example gonad tissue

Gonad stage before pH treatment (n=54) and after 52 days in ambient pH (7.82±0.02, n=39) and low pH (7.31±0.02, n=39) for all populations combined. Gonad differed significantly between pH treatments (𝝌2=9.79, p=0.032), and between pre-treatment and ambient pH (𝝌2=6.61, p=0.146)


Shelly’s Notebook: Mon. Jan 28, 2019, Geoduck Broodstock DNA extraction from Hemocytes

Kaitlyn and I isolated DNA from four Jan. 4 hemocyte samples. Two samples were from Tank 2 (low pH) and two were from Tank 3 (ambient) treated animals. We used the EZNA kit and followed the manual with the following exceptions:

  • We started on step 2 because we had cells not tissue, so no tissue homogenization was necessary
  • Heat block in rm 213 was 59-65C instead of 60C because the digital setting was a few degrees off from the alcohol thermometer.
  • In step 6, the volumes transferred were:
    • 250uL for sample 16
    • 300uL for sample 15
    • 350uL for sample 26
    • 400uL for sample 25
  • We eluted with 2 x 50uL elution buffer warmed to 70C.

Then used 1 uL of each sample for Qubit BR.

Standard 2 read 98 ng/uL and 102 ng/uL, so average is 100 ng/uL which is what it should be.

Here are the following qubit readings for the samples:

Animal ID Tank Treatment Conc. (ng/ul) Total DNA (ng)
15 3 amb 12.3 1230
16 3 amb 56 5600
025 2 low (pH 6.8) 8.3 830
026 2 low (pH 6.8) 7.4 740

We will send these for WGBS sequencing.

from shellytrigg

Grace’s Notebook: Qiagen RNeasy Micro Plus Kit with Geoduck hemolymph and oyster tissue

Today I also tried the Qiagen RNeasy Micro Plus Kit on geoduck (P. generosa) hemolymph (2 samples) and oyster (C. viriginica) tissue (2 samples).

What I did today:

Samples used:

The geoduck hemolymph (1H and 2H) are the same from what I used in my Trizol LS Reagent test. I used 250ul of each sample for the Qiagen RNeasy Extraction.

The oyster tissue samples 0120 and 0139 are from Yaamini’s DNR project. I used the whole tissue sample, and after adding the 2-ME and Buffer RLT mixture, I homogenized the samples using blue plastic mini pestles.

Protocol: page 1; page 2

I used 10ul of 2-ME per sample, with 1mL of Buffer ATL in step 1.

During steps 5-7, I centrifuged at 10,000 rcf for 30s. Tube 0139 (oyster tissue) always had some extra liquid in the spin column, but it all got spun through after the 2min centrifuge at 10,000rcf after step 7.

Eluted samples with 14ul of RNase-free water (provided in kit).

Qubit results (used 1ul of sample):

The geoduck hemolymph samples were “Out of range TOO LOW”. Here’s what the graphs looked like for both hemolymph samples:

The oyster tissue samples were “Out of range TOO HIGH”. Here’s what the graphs looked like for both tissue samples:

Next steps:

Not sure if I should spend the time to bioanalyze the oyster tissue samples from this extraction as well as from the Trizol LS Reagent Extraction

I’ll create an issue to see what next steps should be.

But it could just be that there wasn’t geoduck hemolymph in the samples I used today. I used the same sample tube for both extractions… The other one she offered had much too small of an amount of hemolymph for me to use for either extraction.

from Grace’s Lab Notebook

Grace’s Notebook: Trizol LS Reagent RNA extraction using Geoduck hemolymph and oyster tissue

Today I tried out the Trizol LS Reagent RNA extraction protocol on two geoduck (P. generosa) hemolymph and two oyster (C. virginica) tissue samples. (GitHub Issue #533)

What I did today:

The samples I used (from this GitHub Issue):

The DNR samples are the oyster tissue samples (0114 and 0116) from Yaamini’s old project.
The geoduck hemolymph samples (1H and 2H) are from when Shelly was trying things out with some geoduck that were kept here at SAFS.

I followed the same protocol as I did for the crab hemolymph, with a few modifications.

After adding the Trizol LS Reagent to the samples and centrifuging them, there was no separation into a supernatant. They all looked like this:

The crab samples looked like this after that same step:

So, instead of transfering any clear supernatant to a new tube (there wasn’t any for these samples), I just moved on and added 200ul of chloroform to each sample, vortexed, let incubate at room T for a couple mins, then centrifuged.

After the samples were left to incubate after the addition of chloroform, there was already some separation:

After centrifuge step with the chloroform they looked like this:
Oyster tissue:
Geoduck hemolymph:

After the addition of isopropanol, incubate at room T, and centrifuge, they all looked like this (not an obvious white gel-like pellet at the bottom… but I continued as though there were one):

The rest of the protocol was pretty much the same, except that I forgot to heat up the heat block earlier, so they waited at room temp for about 5 mins before they incubated at 55C.

Qubit Results (google sheet link):

There was no detectable RNA in either of the geoduck hemolymph samples.

There WAS a lot of detectable RNA in one oyster tissue sample, and too much in the other. The Qubit screen was this for the one that was too high:

Next move:

Try out the geoduck hemolymph and some oyster tissue samples using the Qiagen RNeasy Plus Micro Kit

Bioanalyze all samples that had detectable RNA based on Qubit results

from Grace’s Lab Notebook

Ronit’s Notebook: qPCRs and Data Analysis

I ran SOD and ATP Synthetase qPCRs last week. Currently, I’m working on getting box plots worked out with the R script–the main issue I’m trying to sort out is how to account for the N/A values for some of the samples as the current plots just ignore it. I’m also adding a statistical analysis portion to the script so that we can start visualizing which treatments are statistically different.

Link to ATP Synthetase qPCR data:

Link to SOD qPCR data:

Grace’s Notebook: Trizol LS Reagent RNA Extraction Attempt

Today I tried the TrizolLS Reagent extraction again, but added a vortex (5s) step after the addition of chloroform to the samples. Throughout the extraction, the samples looked pretty similar to how they did during my last attempt, and the Qubit results were the same: “out of range”.

Here’s the protocol of what I did today:

Trizol LS Reagent Extraction Protocol:

Lyse samples and separate phases:

  1. Add 750uL Trizol LS Reagent to the samples (pelleted C. bairdi hemolymph)
    (Need to maintain a 3:1 ratio of Trizol to sample. I estimated that the samples are about 250ul)
  2. Homogenize sample by pipetting (5x per sample)
  3. Centrifuge 5 mins at 12000g at 4C
  4. Transfer clear supernatant to a new, labeled tube (The pink Trizol should be at the bottom, but in both attempts, the Trizol was on top, so I had to pipet it out to get to the clear “super”-natant)
  5. Incubate 5 mins to permit complete dissociation of the nucleoproteins complex.
  6. Add 200ul of chloroform and close lid
  7. Vortex 5s
  8. Incubate at room T 2-3 mins
  9. Centrifuge 15mins at 12000g at 4C
    (The mixture separates into a lower phenol-chloroform, and interphase, and a colorless upper aqueous phase)
  10. Transfer aqueous phase containing RNA to a new tube by angling at 45˚ and pipetting out.
    (DO NOT transfer any of inerphase or organic layer. ONLY the aqueous phase)

Isolate RNA

A. Precipitate RNA

  1. Add 500ul of ispropanol to the aqueous phase
  2. Incubate at room T 10 mins
  3. Centrifuge 10 mins at 12000g at 4C
    (Total RNA becomes a white gel-like pellet at the bottom of the tube. In both attempts, it was hard to see. It was never white – always clear at the bottom, so I just continued with the protocol as though there were a gel-like pellet at the bottom.)
  4. Discard supernatant

B. Wash the RNA

  1. Resuspend pellet in 1mL of 75% ethanol
  2. Vortex briefly. Centrifuge for 5 mins at 7500g at 4C
  3. Discard supernatant
  4. Air dry pellet 10 mins (This part was challenging in both attempts because I couldn’t see any pellet at the bottom.)

C. Solubilize the RNA

  1. Resuspend pellet in 20ul of 0.1% DEPC-treated H20
  2. Incubate in a heat block set to 55˚C for 15mins

Run 1ul of each sample on Qubit

Results: All three tubes were “out of range”

Next steps thoughts:

  • Go back and try the Qiagen Kit again
  • Maybe try the kit or Trizol LS with other hemolymph samples from other animals? Or maybe tissue samples from other animals? Just to see if the process works? (A thought that Shelly had while we were talking today)

from Grace’s Lab Notebook

Sam’s Notebook: DNA Isolation and Quantification – Ronit’s C.gigas Ploidy Ctenidia

Last week, I isolated DNA from all of Ronti’s ctenidia samples, however one sample (D18-C) didn’t isolate properly. So, I performed another isolation procedure with another section of frozen tissue. Tissue was excised from frozen tissue block via razor blade (weight not recorded) and pulverized under liquid nitrogen. Samples were incubated O/N @ 37oC (heating block) in 350uL of MB1 Buffer + 25uL Proteinase K, per the E.Z.N.A. Mollusc DNA Kit (Omega) instructions.

After the O/N incubation, I processed the samples according to the E.Z.N.A. Mollusc DNA Kit (Omega) with the following notes:

Sample was eluted in 150uL of Elution Buffer.

Sample was stored in Ronit’s gDNA Box #1 (position B9) (Google Sheet).

I then thawed all the other ctenidia samples isolated last week and quantified them using the Roberts Lab Qubit3.0, using the Qubit dsDNA BR Kit (Invitrogen). I used 1uL of each sample.