Grace’s Notebook: Dia Analysis Re Do

I re-did the DIA analysis from the beginning. This time I only used settings used by Emma in her Skyline document that she sent me.

I think I must have done something better this time around, because a lot of the chromatograms are looking much better. Now I will do the error rate calculation and finally get into the data analysis after that.


Error rate assessment

Skyline doesn’t always do the best job of identifying peaks (where transitions align, meaning that the peptide is real and can be identified). So, error rate has to be calculated.

In order to do this, I will look at ~100 randomly selected peptides and give them a rating of “1” or “0”.

A rating of “1” will only be applied if the following three conditions are met:

  • The peak selected is likely a real peptide (either has an ID and/or the transitions align well)
  • The peak boundaries encompass the peak well
  • The same peak is at about the same retention time and is selected across all replicates (in my case, the four replicates)

A rating of “0” will be given if not all three conditions are met.

I’m not sure if I should figure out a way to identify which condition wasn’t met.



Yaamini’s Notebook: Gonad Histology Update 4

Another classification revision

I met with Brent last week to go through my histology classifications. He recommended that I review the some specimens with the microscope.

Retaking histology images

I read another paper he recomended, Coe 1932, to understand the differences between primary ovogonia and spermatagonia. I read the paper, as well as some guides on tissue types from Carolyn Friedman, then had some dedicated microscope time. Here are the revised classifications and some notes. The classifiation spreadsheet can be found here, and the new images I took can be found here for pre-experiment sampling and here for post-experiment sampling. Shoutout to Grace for helping me set up the iPhone + microscope contraption!

  • Gigas_02: Stage 1 Female. Need to verify that it’s not male. Ovogonia are closer to the walls of the acini, so I’m pretty sure it’s female
  • Gigas_04: Same as above
  • Gigas_05: Looking at the primary sex cells, they’re farther away from the walls of the acini. This may be male? There’s also a chance that I didn’t find any acini and I’m looking at the digestive gland or intestines instead. Need to clarify with Brent.
  • Gigas_06: Stage 1 Female
  • Gigas_07: No acini structure, so Stage 0
  • Gigas_08: Same as above
  • Gigas_10: Same as above
  • Gigas_15: Found some spermatazoa! No acini structure, so it’s a spent oyster. Stage 4 Male!
  • Gigas_18: No acini structure, so Stage 0
  • Gigas_20: Same as above
  • 4-T3: Same as above
  • 5-T3: Same as above
  • 9-T2: Same as above
  • 10-T3: Same as above
  • 12-T6: Spermatazoa but no acini structure. Stage 4 Male
  • UK-03: No acini structure, so Stage 0
  • UK-05: Spermatazoa but no acini structure. Stage 4 Male

Gonad maturation analyses

Not much changed from my previous analysis. Sex is still the only factor that explains differences in maturation. However, I now have different mature and immature classifications between treatments. None of my low pH animals were mature. In my ambient pH treatment, six individuals were immature and four were mature. When I was building my binomial GLM using stepwise regression methods, I got a p-value of 0.03 when I had a Mature ~ Treatment model. However, Sex was a more significant factor, so I added that in first to create my base model. When I used add1 to identify which covariates to add, none of them were significant! Guess I don’t have to change the story in my NSA presentation.

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Grace’s Notebook: Dia Rna Isolation Has Begun

DIA Analysis

Emma came over and took a look at my 2015 Oysterseed Skyline DIA Analysis file. She said that it doesn’t look right and that I should re-do it and make sure I’m following Laura and Yaamini’s settings and/or comparing it to the Skyline document that she sent me.I looked at it a little today, but am just very confused. I have looked at the tutorial from SKyline, and followed the protocols, but I still don’t understand what’s going on. I’ll give it another go tomorrow morning before lab meeting.

RNA Isolation

Today I started isolating RNA. I started out with 9 samples from infected, ambient crabs. Here is the spreadsheet of all the samples that I will use for RNA isolation (if I’m unnsuccessful with any, there are many other samples I could use).



Grace’s Notebook: Rna Isolation Sample Picking Podcast

RNA Isolation Sample picking

I picked some samples for RNA Isolation. I will begin tomorrow! Here is the link to the spreadsheet with the selected samples: here


I created a little podcast intro for DecaPod. I also edited the meeting recording from when Steven and I talked last week. I don’t know how to publish them to the bitter crab website, so I’ll ask Steven tomorrow. I want to record a background information podcast episode for next week.

DIA Analysis

Emma had to reschedule our meeting today for tomrorrow. But she will help me understand how to calculate the error rate more accurately, becasue I don’t think I know what I’m doing!


Laura’s Notebook: DNA Isolation from frozen Oly larvae

It is happening. My trial last week worked sufficiently well in the samples whic started with ~30ug tissue, so I’m going to move forward with the actual extraction. As per Sam’s suggestion, I read the MethylMiner kit instructions to see what finished product I’ll need for the DNA methylation enrichment step. Here’s what I learned:

  • The kit provides materials for 25 affinity-based separations when starting with 5 ng–1 μg of fragmented genomic input DNA, and is scalable up to a single separatation using 25 μg of input DNA. The methlyated DNA may be eluted into as many as 8 fractions per separation.
  • For downstream analyses like PCR and qPCR, as little as 5 ng of input DNA can be used. For applications that require larger amounts of methylated DNA, such as library construction for high-throughput sequencing or amplification and labeling for microarray analysis, starting amounts of 10–25 μg of fragmented input DNA are most appropriate, though in some cases as little as 1 μg can be used. Typical total yields of mammalian CpG-methylated DNA are 3–20% of the input mass of DNA, or 0.3–5.0 μg when starting with 10–25 μg.
  • DNA may be fragmented using your method of choice. DNA must be fragmented to an average size of less than 1,000 bp and should be in DNasefree water, TE buffer, or another low ionic-strength, neutral pH buffer. The fragment size should be appropriate for your downstream analysis. For example, DNA fragmented to an average length of ~250 bp is suitable for assay by real-time quantitative PCR (qPCR). Similarly, DNA fragmented to an average length of ~100–200 bp is suitable for fragment library construction for short-read high-throughput sequencing.
  • For input amounts <1ug, add 5 ng–1 μg, added volume should be ≤ 80 μl
  • For input amounts 1 μg-10μg, the fragmented DNA should be at a concentration of 25 ng/μl or higher; added volume will be 40–400 μl.
  • For input amounts 10-25ug, the fragmented DNA should be at a concentration of 25 ng/μl; added volume will be 400–1000 μl.

Since I need to shoot for at least 10ug DNA per sample (which is a lot), I will do the following:

  • Increase my initial tissue mass to 50mg, which is the max I can do for each column.
  • Extract 3 replicates from each tissue sample; I should have plenty of larvae in each sample to do so.
  • I should get ~3ug DNA per sample, given the yield from test run.
  • For final elution volume, I should not exceed 300uL.

from LabNotebook

Laura’s Notebook: Oly DNA Isolation, take 3

Soaked mortar, pestle, metal spatulas in 10% bleach/DI water (100mL Clorox bleach, 900mL DI water) for ~15-20 minutes. Rinsed with DI water, let dry then covered individually in foil and autoclaved at 121C for 20 minutes.

Next day, put mortar & pestles in -20 for a copule hours. Picked up 6L of liquid nitrogen – note this is availabe in the Bagley, the Chem supply store, not in the J-wing of the microbiology building where we get dry ice.

Got larvae and mantle tissue out of the -80 for my test run, on the following groups:

test10 27-A HL-10-Low Frozen larvae
test11 60-A HL-10-Low Frozen larvae
test12 73-A HL-10-Low Frozen larvae
test13 59-A HL-6-Ambient Frozen larvae
test14 HL-10-12 HL-10-Low Frozen mantle
test15 HL-10-10 HL-10-Low Frozen mantle
test16 HL-6-18 HL-6-Ambient Frozen mantle
test17 HL-6-20 HL-6-Ambient Frozen mantle

Used E.Z.N.A. Mollusc DNA Kit NOTE: we have 8 mortar/pestle sets in our lab. Can’t do more than 8 samples / day.

Labeled and weight 1.5mL microcentrifuge tubes.

Held samples on wet ice until homogenized. For larvae, they were all at the bottom of the tube. I inverted the tube and tapped vigorously to get as much onto the mortar as possible; also used autoclaved toothpicks to try to get more out. Probably shouldn’t do this next time – find another small item to scrape tissue out of tube. Curious if I can add ethanol or something to get the larvae into solution, then pour into mortar? Will ask the lab.

Poured LN2 into mortar with pestle also in; grouned tissue down. Used metal spatula to scrape frozen tissue dust into pile, carefully transferred into new 1.5mL microcentrifuge tube. This was also very difficult, as the spatula just barely fit into the tube, and if the tissue started to melt at all it would stick to the spatula. Need a better tool for transferring tissue – smaller spatula with round tip? Also, sterilized the spatula by soaking in bleach, then 2 DI rinses, between every-other sample (used 2 spatulas). Adequate? Will ask lab.

Need no more than 30mg for each sample; weighed tubes+samples. For samples with >30mg, labeled and weighed new tubes and either transferred ~30mg into newly labeled tube or removed a bit of sample.

Added 350uL buffer, and 25uL proteinase K. Floated in 60C water bath. Kit says to incubate for 30 mins – 4 hours.


from LabNotebook

Grace’s Notebook: Updates Podcast Dia


Today Steven and I recorded a little bit of us talking about the spreadsheet and how I’ll be doing the RNA isolating. What I am going to work on is an episode, or several short episodes, about the background of the project: bitter crab disease; Tanner crabs; Juneau, etc. I also am going to make a quick little intro that we can put at the beginning of all the episodes. I want to make one weekly, or at least release one weekly. And I would like to have some way to make it more easily understandable to people like my mom, or my friends. To do that I think I just have to make sure I explain what I’m talking about more. Obvoiusly I shouldn’t be putting too much time into it because the main focus is on the research itself, but I’m excited and looking forward to learning more about this outreach tool and communication strategy.

DIA Analysis

I looked at the DIA analysis of the 2015 oysterseed. I have no idea what I’m looking at anymore and am getting confused. I read a Skyline tutorial that Emma recommended I look at in order to learn more about how to tell if Skyline is picking peaks well (tutorial) but I am not fully grasping it. So, Emma is going to stop by on Monday to show me what’s what!

RNA Isolation

I am going to pick samples such that I’m following individuals throughout the entire project and within treatment groups. I’ll begin next week and will keep track of what I’m doing.

Crab data sheet

I re-organized the data sheet such that it is machine-readabe. Per Steven’s suggestion, I made a unique ID for each sample following this pattern:


Example: 6116_76_9

6116 is the unique crab ID number (there are three samples with 6116_ at the start, because this data includes only crabs that survived the project, and thus have three samples.

76 is the tube number that the hemolymph sample is in.

9 is the sample day number.