# 72 anemones each in 200mL volume... how much volume I will need to change every other day
ml <- 72*200
print(ml)[1] 14400# how much of each treatment (seawater vs. leachate)
ml/2[1] 7200September 2024
03-SEP-2024
Received confirmation that RNA is in line for QAQC
Read manual for Nikon compound scope, setup for making embryology preservation slides & taking photos
02-SEP-2024
Submitted Sample Form to Azenta
Writing & editing chapter 1 methods
August 2024
28-AUG-2024
- Received email confirmation of receipt of RNA at Azenta!
27-AUG-2024
Shipped Total RNA to Azenta in 96 well plate on ~16lbs of dry ice, FedEx Priority Overnight from SeaTac FedEx Office Ship Center
Tracking #: 278809181237
15-AUG-2024
Met with Jefferey Kim of Azenta GeneWiz
Got a quote for 60 TotalRNA-seq with PolyA selection, 20M reads per sample
For Total RNA in nuclease-free water on dry ice, need 100ng of Total RNA… so if I have 10ng/uL in 30 uL… that means there is 300 ng of Total RNA in that sample. Jeff said 10ng/uL is usually the cutoff. I can send Total eluted RNA in 30uL without normalizing or Nanodropping… they will run RIN scores when they receive the samples. I just have to extract and Qubit!
23-28 business days turnaround time
With discount, price per sample is $98.50! Total cost is $5,910.00 in quote :D
March 2024
28-MAR-2024
met with Kip and made calendar of assignments due
changed out filters on seawater filter rack in PBB
To measure impacts of microplastic exposure on anemone size, oral disk diameters were calculated using the average of two perpendicular measurements across the oral surface intersecting at the mouth. Measurements were made using Image J software (Rasband 19972018) with digital photographs of each anemone …. - Beck, 2022 WWU Dissertation
How do we get scaled images of the oral disc?
downloaded AmScope software for MU503B that is attached to the dissecting scope in PBB: https://amscope.com/pages/software-downloads
downloaded AmScope manual: https://storage.googleapis.com/software-download-d79bb.appspot.com/Manual%20Download%20Files/Manual%20Files/Cameras/MU_MD_Series_Camera_Software_Manual.pdf
21-MAR-2024
spent nearly all day drying an edge of each petri dish, and gluing bee-tags to them with superglue, then letting it dry by floating the petri dish on a floating rack in the seatable. Took all day..
swapped anemones from upper seatable to lower, changed water in upper, and letting it come down to temp (when doing water changes, cannot do it directly, as header water is at room temp and seatables are at 10C…. How will I deal with this during the experiment when each anemone is in a 200mL jar and I’ll be doing water changes every other day
The amount of microplastic may be our limiting factor for how long the exposure is.
We have 3 grams of each microplastic type
To make a batch of leachate according to the Saliu Lab protocol, we have 250 mg of microplastic.
# 3 grams is 3000 mg, how many batches of leachate will each microplastic type make?
3000/250[1] 1212 batches… that’s a lot, and should be plenty since it is a highly concentrated amount that will be diluted.
How will it be diluted?
20-MAR-2024
- cleaned anemones by picking rocks and debris from them, and squirting with chilled seawater from a lab squeezie bottle. Two were ‘escapees’, one was fully out of the petri dish! I popped it back in. The ones that are nudging around the edge/lip of the petri dish I use my finger to just push their pedal disc back inside the dish… not sure what I can use to deter them from escape that won’t expose them to more chemicals (i.e. tape ahdesive, etc.)
February 2024
22-FEB-2024
Roberts Lab meeting, received feedback on anemone research proposal: suggested I do initial response curves to find at what temp A. elegantissima experience stress, at what microplastic leachate concentration do they experience stress? Measure response variables ‘quickly’ by respirometry and PAM. Can I make the single stress, multiple response variable, the first chapter of the Master’s thesis? Then, two stressors together with `omics response variables (once we find the inflection points of stress) can be the second chapter…. Ariana pointed out that the paper reviewers are going to want to know why I chose certain concentrations of microplastic leachate. I have to justify that by doing a response curve first. Learned about Roberts Lab pubathon.
Took FireSting O2 to IT to try and get correct drivers on
mariscosruggedized laptop to run the program…pyroscience FireStingO2 deemed ‘dead’ by IT specialist Michael Shriver… the unit won’t connect. I sent the following to the Walz product support team Todd Kana (kana@bayinstruments.com) & Erich Keim (ekeim@walz.com):
Good afternoon Erich and Todd,
I’m a graduate student researcher in the Padilla-Gamino Lab at the University of Washington, and I’m working with a Walz Mini PAM-II and FireStingO2 oxygen meter that our lab purchased from you as a set. The instrument was previously used by Miranda Roethler, who passed your contact information on to me.
The FireStingO2 oxygen meter is not powering up, and won’t connect to any computer that we’ve tried it on. It was just with our IT specialist for the last few hours (he was attempting to get it to connect by downloading old drivers), and he shared that the laser-cut `FiresStingO2` light on the instrument would ‘pulse’ red and then remain off. He deemed the instrument ‘dead’…
This instrument has only been used a handful of times, and it has been kept in good condition (not dropped, or been exposed/submerged in water etc.).
Have you had this issue before? If so, are there any solutions you can help provide?
Kindy,
- Sarah Tanja
21-FEB-2024
pyroscience FireStingO2 only works on Windows 10!?
Ammonia levels in PBB header tank are at 1ppm, something green is growing in there. When was the last time we checked that the UV sterilizer was working? I should change out all the filters on that seawater purification rig, and the reverse osmosis deionized water filter cascade.
There is algae growing in the PBB header/holding tank
Met with Kip, Added Kip to WDFW scientific collection permit
9 permit locations!
Messed around with mapping the collection sites, from R Graph Gallery, https://r-graph-gallery.com/180-change-background-in-leaflet-map.html
All map background by leaflet can be found here: https://leaflet-extras.github.io/leaflet-providers/preview/index.html
Interactive Map with leaflet
## set CRAN mirror
options(repos = c(CRAN = "https://cran.r-project.org/"))## install packages
if (!require("tidyverse", character.only = TRUE)) { install.packages("tidyverse")}Loading required package: tidyverseWarning: package 'tidyverse' was built under R version 4.2.3Warning: package 'ggplot2' was built under R version 4.2.3Warning: package 'tidyr' was built under R version 4.2.3Warning: package 'readr' was built under R version 4.2.3Warning: package 'purrr' was built under R version 4.2.3Warning: package 'dplyr' was built under R version 4.2.3Warning: package 'stringr' was built under R version 4.2.3Warning: package 'forcats' was built under R version 4.2.3Warning: package 'lubridate' was built under R version 4.2.3── Attaching core tidyverse packages ──────────────────────── tidyverse 2.0.0 ──
✔ dplyr 1.1.4 ✔ readr 2.1.5
✔ forcats 1.0.0 ✔ stringr 1.5.1
✔ ggplot2 3.5.1 ✔ tibble 3.2.1
✔ lubridate 1.9.3 ✔ tidyr 1.3.1
✔ purrr 1.0.2 ── Conflicts ────────────────────────────────────────── tidyverse_conflicts() ──
✖ dplyr::filter() masks stats::filter()
✖ dplyr::lag() masks stats::lag()
ℹ Use the conflicted package (<http://conflicted.r-lib.org/>) to force all conflicts to become errorsif (!require("leaflet", character.only = TRUE)){ install.packages("leaflet")}Loading required package: leafletWarning: package 'leaflet' was built under R version 4.2.3## load libraries
library(tidyverse)
library(leaflet)grey <- leaflet() %>%
addTiles() %>%
setView( lng = -122.8, lat = 47.9, zoom = 8 ) %>%
addProviderTiles("Esri.WorldGrayCanvas")
greyterrain <- leaflet() %>%
addTiles() %>%
setView( lng = -122.8, lat = 47.9, zoom = 8 ) %>%
addProviderTiles("Esri.WorldTerrain")
terrainStadia.AlidadeSmooth <- leaflet() %>%
addTiles() %>%
setView( lng = -122.8, lat = 47.9, zoom = 8 ) %>%
addProviderTiles("Stadia.AlidadeSmooth")
Stadia.AlidadeSmoothStadia.OSMBright <- leaflet() %>%
addTiles() %>%
setView( lng = -122.8, lat = 47.9, zoom = 8 ) %>%
addProviderTiles("Stadia.OSMBright")
Stadia.OSMBrightJawg.Lagoon <- leaflet() %>%
addTiles() %>%
setView( lng = -122.8, lat = 47.9, zoom = 8 ) %>%
addProviderTiles("Jawg.Lagoon")
Jawg.LagoonStadia.StamenWatercolor <- leaflet() %>%
addTiles() %>%
setView( lng = -122.8, lat = 47.9, zoom = 8 ) %>%
addProviderTiles("Stadia.StamenWatercolor")
Stadia.StamenWatercoloremailed Hannah at Seattle Aquarium about seawater pickup for Friday FEB 23 from 10-4
reserved SAFS Truck for seawater pickup
20-FEB-2024
Downloaded pyroscience FireStingO2 Pyro Oxygen Logger
First tried on my computer Owl, USB was not recognizing, so we’re trying it on QuelpoToo
The USB connection doesn’t connect to QuelpoToo either (is it the cable?)
02-FEB-2024
Follow-up with UW office of veterans affairs about debt
Lab meeting notes:
- move shaker table to lab 242 hood
- move nitrogen needle lipid extractor to methanol/chloroform cabinet in 242
- ok to buy organizational bins for PBB, keep receipts & get reimbursed
- respirometry chamber battery operation problematic… reduced flow, battery drain over time (30mins); Nuria left a plug-in chamber somewhere in PBB
Writing your resume for federal job webinar
schedule A: disability accommodation
Federal hiring resume, there is no page limit
The resume gets you to the hiring manager, show in the resume that you qualify before it goes to a hiring manager
hiring managers in Federal hiring do not click on links (github, linkedIn, etc.) they won’t be viewed…
‘Your resume is the movie trailer…’ Highlight the level of complexity of your work
Itemize how how manage something, on top of listing your role… you have to explain what you did in that role. The role ‘president of xx’ doesn’t translate to the person reviewing the resume knowing what you can do… leave nothing to the imagination. Be explicit.
Spoken presentations: how many presentations you have given, what & when
The first person that looks at your resume is a human resources specialist, not a scientist!
show that you are an ‘innovator’ and that when you see a problem, you take initiative to fix it.
where you go, people are impacted positively, there is recognition, things change or improve, things get better, you save money, you meet deadlines, you bring about conflict resolution.
the cover letter is not used to qualify you, ONLY THE RESUME!
Vet team to assist navigating federal hiring process: fedshirevets@opm.gov
You get shuttled into qualified, well-qualified, best-qualified. Qualifying doesn’t mean that you get referred to the hiring manager… you can qualify but not get referred to the hiring manager. You can be referred, but not get an interview.
wrp workforce recruitment program
08-FEB-2024
attended Roberts Lab meeting
- Will be presenting experiment design & proposal on Feb 22nd
worked on dropbox-uploader app in raven server to try and get data from dropbox directly to raven *not working
07-FEB-2024
Identified anemones from Alki Beach have Symbiodinium muscatinei
Identified anemones from Teddy Bear Cove have Symbiodinium muscatinei
05-FEB-2024
Lab meeting cancelled
emailed Francesco Saliu asking about sending their lab SPME fibers for analysis
- still no package or protocol, starting to think about plan B/C/D…etc
found rnoaa package and zenodo by Wendell Raymond https://zenodo.org/records/6555076 from paper “Assessment of the impacts of an unprecedented heatwave on intertidal shellfish of the Salish Sea” recommended by Bob Oxborrow
- explored rnoaa package
13:30 - shadow fish 427 lab on photosynthesis and respiration with Miranda
Goal for the week:
- Get PAM photosynthesis initial data
January 2024
December 2023
November 2023
27-NOV-2023
- Lab Meeting:
- Requested to order cultured abalone sea urchins
- Reminded JPG about meeting with Alison Gardell Tues Nov 28th at 1:30PM via Zoom
- JPG requested I put together slides for meeting with Alison Gardell
- Got seawater from Seattle Aquarium
- Met with Kip and got them setup with safety trainings
- Must order sea urchins on Mon Dec 11th for a Mon Dec 18th/Tues Dec 19th delivery
21-NOV-2023
2nd Master’s Thesis Committee Meeting
- Trying to come up with a solution to lack of solvent control.. how to move forward
11-NOV-2023
- WSN day 4
10-NOV-2023
- WSN day 3
09-NOV 2023
- WSN day 2
08-NOV-2023
- WSN day 1
05-NOV-2023
Undoing large file commit on coral-pae-temp for ~2hrs 🤬, tried to just undo the one commit, but couldn’t get it to work. Had to execute
git reset –hard origin/mainAdded the following to the
git/hooks/pre-commitfile:#!/bin/bash echo "automatically ignoring large files" find . -size 5M | sed 's|^\./||g' >> .gitignore cat .gitignore | sort | uniq > .gitignore git diff --exit-code .gitignore exit_status=$? if [ $exit_status -eq 1 ] then set +e for i in `cat .gitignore` do set +e git rm --cached $i done git add .gitignore git commit .gitignore --no-verify -m"ignoring large files" echo "ignored new large files" fi
03-NOV-2023
Finished extraction, nanodrop & qubit for 12 samples
6MH1a
10OH1a - greenish, bad! no DNA/RNA, degraded!
6PA2a
5HH1a
5HA1a
9OA2a
7HA1a
3CA1a
8CA1a
2OA1a
2MH1a
5CA2a
02-NOV-2023
Started extraction of 12 samples, paused after adding 350uL of DNA/RNA Lysis Buffer to 350uL of PK-Digested sample supernatent, and frozen in -80 freezer:
- 6MH1a
- 10OH1a - green
- 6PA2a
- 5HH1a
- 5HA1a
- 9OA2a
- 7HA1a
- 3CA1a
- 8CA1a
- 2OA1a
- 2MH1a
- 5CA2a - green
Finished qubit of 10 samples:
1MH2a
2LA1a
1MA1a
1OH2a
6PH2a
10LH1a
9LA2a
8CH2a
8OA1a
5OH1a
01-NOV-2023
Finished extraction & nanodrop of 10 samples:
- 1MH2a
- 2LA1a
- 1MA1a
- 1OH2a
- 6PH2a
- 10LH1a
- 9LA2a
- 8CH2a
- 8OA1a
- 5OH1a
GSS Presentation Design Workshop with Tim Essington
Presentations should have ONE THING they are communicating: ‘Here’s a nugget’
One Word Theme
One Sentence Summary
Add motion to a few slides to break things up visually
Use a blank slide and pause at a key pivot in your story
build the slide iteratively when you have a complex figure
story arc:
ordinary world
challenge
series of actions to overcome challenge
resolution
new world
‘Lean into your ’buts’
leave silence + space between story arcs
October 2023
27-OCT-2023
QA/QC’d samples from 26-OCT-2023
Met with Kip & moved experiment setup to PBB
Collected glycerol from Jon Bakker at Horticultural Center Merill Hall
26-OCT-2023
extracted samples:
6LH1a
8LA2a
10CH2a
5CH2a
4HA2a
7OA2a
2MA2a
3LH1a
5LA1a
8MH2a
24-OCT-2023
- working on WSN presentation!
23-OCT-2023
JPG 1on1
Bought Amtrak tickets to WSN (refundable)
16-OCT-2023
- attended lab meeting via Zoom
“Total RNA was extracted using the Duet DNA/RNA Miniprep Plus Kit (Zymo Research, Irvine, CA, USA, Cat #D7003) with modifications to the manufacturer’s sample preparation steps, as described here. Sterile clippers were used to remove an approximately 1-cm diameter region from the coral tissue. The tissue clipping was placed in 500 μL of DNA/RNA shield (Zymo Cat #R1100-50) with 0.5 mm glass beads (Fisher Scientific Cat #15-340-152) and homogenized by vortexing for 1 minute. The supernatant (450 μl) was transferred to a new tube and centrifuged at 9000 rcf for 3 min to remove any debris. The supernatant (300 μl) was mixed with 30 μl of Proteinase K (Zymo Cat #D3001-2-20) digestion buffer and 15 μl of Proteinase K in a new tube using a brief vortex step. Equal volume of Zymo lysis buffer (Zymo Cat #D7001-1-200) was added (345 μl) and extraction was completed as outlined in the manufacturer’s protocol. RNA concentration was quantified using the Qubit RNA Broad Range kit (Invitrogen Cat #Q10211) and integrity was assayed with an Agilent TapeStation 4200 system and the RNA Integrity Number (RIN) values ranged from 7.0–8.9. RNA libraries were prepared using the Zymo-Seq RiboFree Total RNA Library Kit (Zymo Cat #R3000) according to the manufacturer’s instructions, starting with 125 ng of RNA as input. Prepared libraries were sent to Zymo Research Corporation for sequencing using the Illumina HiSeq for paired end sequencing (2 × 100 base pairs [bp]).” - Wong & Putnam 2022 (Wong and Putnam 2022)
Wong, Kevin H., and Hollie M. Putnam. 2022. “The Genome of the Mustard Hill Coral, Porites Astreoides.” Gigabyte 2022 (July): 1–12. https://doi.org/10.46471/gigabyte.65.
11-OCT-2023
Attended ISB workshop day 1
Gibbons-Lab github repo: https://github.com/Gibbons-Lab/isb_course_2023/tree/main
Course presentation slides: https://gibbons-lab.github.io/isb_course_2023/16S/
Multinomial Naive Bayes
qiime feature-classifier classify-sklearntrained on V4 region of the 16S marker genecenter log ratio transform abundance counts
recommended papers:
Microbiome differential abundance methods produce different results across 38 datasets
Beta diversity (or Ordination) analysis“PCoA and NMDS methods take the distance matrix as an input and are sensitive to distance method used. The PCoA maximises the linear correlation between samples, wherein NMDS maximises the rank-order correlation between samples. Additionally, in case of NMDS, data is not required to fit a normal distribution.” This link has some good visualizations: https://www.davidzeleny.net/anadat-r/doku.php/en:pcoa_nmds
Why is the mean being used instead of the median or the modal? Bacteria distribution is not a normal distribution across populations…. This is a log ratio of (relative abundance/geometric mean). It’s a common method for transforming compositional data. For sequencing data normalization does not mean making anything normally distributed but rather putting samples on the same scale… Here’s a review on the topic: https://www.nature.com/articles/s41522-020-00160-w
NatureAnalysis of microbial compositions: a review of normalization and differential abundance analysis
10-OCT-2023
- Bought WSN flights
- Sent list of 123 samples to UW mim_c for formal quote
- filled out GI Bill enrollment verification
- updated all extraction notebook posts from handwritten notes
03-OCT-2023
- Submitted abstract to WSN, registered for conference!
02-OCT-2023
Abstratc accepted to SICB for Jan 3rd!
Your abstract Corals in a hot plastic ocean: How heat and phthalates shift a Montipora capitata coral microbiome has been accepted for the 2024 Annual Meeting in Seattle, WA, on 2-6 January 2024. Your abstract id is 1230.
You presentation is in the Response to anthropogenic environmental changes Concurrent session on Wednesday, January 03, 2024.The online program can be found HERE. If you need to make a change to your abstract or cancel it you MUST fill out this FORM. This is the only way changes and cancellations will be accepted.
Information regarding presentations at the SICB meeting can be found HERE.Please note that all presenters must register for the meeting by the presenter registration deadline of 1 November 2023, otherwise your talk may be cancelled. Click HERE to register. Please visit the meeting web site for complete information on registering and conference activities. If you have registration questions email registration@sicb.org, for meeting questions email lstrong@burkinc.com.
September 2023
20-SEP-2023
05-SEP-2023
WSN newsletter says abstracts close Sep 29th 2023
5 min lightning talk or 15 minute talk
Abstract Guidelines
The abstract should briefly introduce the project, the goals, the results, and the significance
The presentation title should have no more than 130 characters max (including spaces) in ALL UPPERCASE
The abstract should have no more than 1600 characters max (including spaces); use standard HTML tags for special formatting (e.g., \<b\>**bold text**\</b\>, \<i\>*italic text*\</i\>, etc.)01-SEP-2023
crashed my bike and badly injured my hands… can’t do lab work 😭
met with UW mim_c Steven Salipantes
I need to send him a sample list for a final quote, but should be $2K for ~100-120 samples (cost of sequencing lane + reagents) .. averages to ~$20/sample, much better than Mr. DNA ~$85/sample, plus no shipping charges.
bring all 100-120 samples together
1.5mL microcentrifuge tubes
50uL volume , 20ng/uL quantity concentration
August 2023
31-AUG-2023
purchased additional kits, qubit assays, tubes, etc. to spend down Hall Grant
received Qubit and UV-C Sterilizer!
extracted:
mock
blank
4-LA2a
1-HH2a
2-HH2a
3-HA2a
3-MA2a
1-Ha2a
4-MA2a
3-HA2a
29-AUG-2023
submitted SICB abstract
submitted Kip’s capstone proposal to Greg Jensen
28-AUG-2023
Look into the Margaret A. Davidson Graduate Fellowship
Wrote abstract for SICB , Seattle Jan 2-6 … Deadline is Aug 29th!
Met with UW mim_c Kyle Asfahl
balanced Hall budget, have ~$5K left to spend
Michael Shriever reset medusaFTIR and downloaded Nanodrop software to it
Take Jeremy’s samples out of freezer to thaw at 4PM
24-AUG-2023
- Trial with 2 ambient samples of modified extraction protocol and RNA Clean & Concentrator. I did 2 samples because I had a kit that had 2 preps left over. Most of the time spent is in lab bench sterilizing, prepping, labelling, getting ice! Which takes literally a 30 minute hike across campus… But I started at 930AM and finished with Qubit in the FTR Roberts Lab by 4PM. With 2 samples! Extractions take ALL DAY.
17-AUG-2023
Are we sure we want to try TriZol for RNA? It isn’t a phenol-free kit like the Quick DNA/RNA Miniprep, and we have RNA, we just need to purify it. Tomorrow lets run all eluted RNA through RNA Clean & Concentrator, and check it on Nanodrop! LFG.
THE BLANK HAD ‘TOO LOW’ DNA IN QUBIT!
Organized extraction notes (handwritten) for extractions 1-8
Reserved 1 King Bed in Hotel Block for WSN
Applied for WSN Student Travel Grant
How will attending the WSN meeting help you advance your career as a naturalist and/or scientist (75 words max)?
Attending WSN would be my first opportunity to share my Masters thesis with the scientific community. As an undergrad, I had a bad first conference experience where I was ridiculed for not having results in a methods poster. I hope to attend WSN because its reputation as a welcoming environment for early-career scientists is the kind of scientific community I wish to be a part of and perpetuate in my own career.
What are you working on to improve diversity, equity and inclusion in your community (75 words max)? (Examples may relate to personal identify, interest, experience, etc.)
I recently took part of the Identity, Belonging, and Inquiry in Science Program, and received special training to mentor students from diverse backgrounds. My mentee worked hard to complete research that she will present at the upcoming SACNAS conference, and I couldn’t be more proud. The training gave me confidence to take on another mentee, this time a Capstone student. Mentorship is a key part of DEI efforts that I find extremely rewarding.
16-AUG-2023
asked JPG to buy benchtop UV Sterilizer for me & Callum, got approval! Purchased by Kat same day :D
NMDS (non-metric multidimensional scaling) ordination plots
walked with Callum, plan to ask JPG to buy Qubit too!
Made some taxonomy bar plots and heatmap dendrograms… still working to understand what they mean but they don’t look like there is separation between treatments. Haven’t gotten any pvalues yet from the composition analyses…
Wondering if I should break up the Notebook into diversity & composition? It’s getting really long and overwhelming with all the analyses moshed together
Still can’t
quarto renderin Jupyter Lab on raven server, need to figure out how to get those interactive jupyter plots in my notebook!
15-AUG-2023
met with Dr. Amy VanCise about microbiome analysis:
Look into work by WHOI Amy Apprill, Laura Weber, Cynthia Becker
Focus analysis on community composition over diversity statistics
different sampling depths across different samples … subsample the counts without replacement so that each sample has the same number of features
Differential abundance analysis on microbial community is similar to differential gene expression analysis!
Sampling depth is normalizing between samples
Look into getting a UV Sterilizer
Do the analysis without blank, mock, & env samples included… get a clean look at only treatments
Look into R Vegan package
Phred score: spatial, 30 (or 25) is good to keep
03-AUG-2023
Received RNA samples back from UT GSAF
Used PCR Inhibitor Removal Kit on five of the samples (see RNA Woes post here) :
1-CA2a
1-CH2a
1-Ea
1-PA2a
1-PH1a
PCR Inhibitor Removal Kit said to add 50-200uL of RNA, but I only could add 30uL. I took the 5 samples from the 96-well plate and augmented them with all the leftover diluted normalized RNA.
Nanodrop note: blank should result in a spectrum that varies no more than 0.050A in the 1mm path. For this batch I used the Roberts Lab Nanodrop and the blank was -0.009/-0.008 10mm path.
July 2023
20-JUL-2023
RStudio>Project Options> Python> Python interpreter
19-JUL-2023
Emailed Jackie about getting another P1000
Helped Bob Oxborrow with pipettes for stable isotope sample prep
Downloaded QIIME2 to Roberts Lab Raven server for coral-pae-temp repo
Shadowed Miranda to learn how to acid wash glassware in 10% HCl:
Scrub and let soak 24hrs in Alconite bath
Rinse 3X in Ultrapure water & shift to 10% HCl bath, let soak for 24hrs
Remove & rinse 3X in Ultrapure water and let sit in Ultrapure water for 24hrs
Remove & let air dry, cover opening with foil
June 2023
07-JUN-2023
- Shipped DNA to Mr. DNA and RNA to UT GSAF!
06-JUN-2023
- Normalized all 20 RNA samples
05-JUN-2023
Re-normalized 2-CA2a DNA and successfully got it to 19.5, 19.2 :D
Worked all day to normalize RNA with mixed results. In the first batch, 7 out of 20 samples were ‘off’ of the target concentration of 25ng/uL by enough to make me want to redo them (one was way high at 230ng/uL, others were too low at ~10ng/uL)
I first re-ran the qubit (2nd qubit run) and got really poor results, with all the rerun samples too-low, (even the one that was previously 230ng/uL!?)
I then re-normalized the samples and ran them on qubit again (3rd qubit run). In this run 4-PH1b hit the target, the rest were still too low
I re-normalized the samples for a third time (with some increasing concentration modifications) and ran them on qubit for a 4th time. In this run 4-PA2a hit the target, the rest were still too low, and 2-PH2a even got worse despite increasing concentration.
Ended the day unsatisfied with the following samples too low for normalization despite 4 attempts:
2-PH2a - too low
2-CA2a - 15.0, 15.4
1-PA2a - 10.2, 10.0
1-PH1a - 17.6, 17.6
3-CH2a - 15.0, 14.6
4-CA1a - 14.0, 13.0
02-JUN-2023
- Normalized DNA (still have RNA left to do)
- Tested normalized DNA on Qubit BR DNA assay, results overall were good but a smidge lower than my goal
- blank + microbial standard extraction
01-JUN-2023
- Presented Bioinformatics final project for FISH 546
- Attended GSR Meeting
- Attended Seminar/TGIT
May 2023
31-MAY-2023
- wrote
dilutefunction to normalize RNA & DNA in coral-DNA-RNA-lab-extractions GitHub repo:
dilute <- function(C1, C2) {
# Initialize variables
n <- length(C1)
C1 <- C1
V1 <- numeric(n) # Initialize V1 vector
V2 <- numeric(n) # Initialize V2 vector
# Loop through rows
for (i in seq_along(C1)) {
# Check for missing data
if (C1[i] <= 0) {
cat("Skipping row ", i, ": Invalid data\n")
next
}
# Initialize V1 and V2 for the current row
V1_row <- 1
V2_row <- (C1[i] * V1_row) / C2
# Loop until conditions are satisfied
while (!is.na(V1_row) && !is.na(V2_row) && (V1_row < 1 || V2_row < 50)) {
V1_row <- V1_row + 1
V2_row <- (C1[i] * V1_row) / C2
# Break the loop if V1 exceeds a threshold value
if(V1_row > 49) {
cat("Skipping row ", i, ": No valid solution found\n")
next
}
}
# Store optimized V1 and V2 values in the corresponding vectors
V1[i] <- V1_row
V2[i] <- V2_row
}
# Create a data frame with C1 column
result <- data.frame(C1 = C1)
# Add V1 and V2 columns to the result data frame
result$V1 <- V1
result$V2 <- V2
result$C2 <- C1*V1/V2
return(result)
}- SAFS Spring Picnic!
29-MAY-2023
- Worked on Bioinformatics Final Project, RNA expression heat map with Gene Ontology Terms (Protein Functions)
- IBIS Celebration of Nora’s Blog!
25-MAY-2023
- Hosted Dr. Nicola Smith as the GSIS
24-MAY-2023
University of Texas at Austin Genomic Sequencing and Analysis Facility (GSAF):
-
TagSeq: 25ng/ul concentration in greater than or equal to 25ul volume (submitting RNA as low as 10ng/ul or as high as 100ng/ul is acceptable BUT ALL samples MUST be submitted at the same or very similar concentrations)
-
23-MAY-2023
- completed UW driver safety training
- met with Steven Roberts, confirmed that no blank/standard is used for RNA-seq
- emailed with Jesse Zaneveld, learned about Zymo mock microbial community standard and microbial community DNA standards to be used as positive controls for the extraction (community standard cells) and the PCR library prep and sequencing (community standard DNA) to assess bias in each process.
- Put both Zymo standards on purcahsing log, emailed Kat and she said she would buy them tonight with the pcard
- Met with Nora, she has made an awesome quarto blog for the IBIS celebration to showcase what she has learned. We also talked a bit about stats and need to follow up with what to do when your data is not normally distributed (moving from paired T test to Wilcoxon paired-sample T test)
- Tried using
quarto publish gh-pagescommand to publish blog
19-MAY-2023
Ran Qubit on ex7 samples
Extraction #7 (the redo) on samples:
- 3-Eb
- 3-PH1b
- 2-PA2b
- 2-CH1b
- 4-PH1b
- 3-CA1b
found out how to use quarto to do Margin References!
found cool article on Verbatim code chunks in R (want to render the code chunk options and show what engine is being used?)
dry-ice at biochem stockroom is low, have to check that there will be a replenishment next week so I have enough dry ice to ship samples!
18-MAY-2023
- wrote script to normalize eluted RNA/DNA
04-MAY-2023
- received Qubit DNA Broad Range (BR) Assay Kit!
- Gotta get P2 DNA/RNA free tips!
- ran qubit on DNA & RNA, got great results!
April 2023
25-APR-2023
- sorted pipettes with Callum
- submitted Bioinformatics Assignment
- ordered Qubit DNA BR assay kit
- ordered film sealing plate roller
24-APR-2023
sent draft letter for NAH experiential learning fund to JPG on Monday April 24th
emailed Mr.DNA about 20 16S samples
read up on NanoDrop vs. Qubit
reached out to Sam White about Ubuntu/Windows dual-boot computer in lab 236, which is connected to the Thermo NanoDrop 2000c Sam shared:
FYI, if you’re solely using the NanoDrop for quantification (i.e. not worried about 260/280), then I’d highly recommend just relying on the Qubit, with both DNA and RNA. The reason for this is that the NanoDrop will almost always over-“calculate” your sample concentrations (both RNA and DNA). The reason for this is that the NanoDrop is a spectrophotometer and measures light absorbance. With almost all DNA/RNA isolations, there will be residual DNA or RNA in your sample. The NanoDrop cannot distinguish between the two molecules, because they’re both made up of the same material; nucleotides. Nucleotides, regardless of their of their origin will absorb light at an average wavelength of 260nm.Since the Qubit assays use molecule-specific dyes, the dye will only bind to DNA or only to RNA. The dyes will not bind to any carryover of other types of molecules in the sample, so the accuracy (and sensitivity) is much, much higher than the NanoDrop.
21-APR-2023
met with JPG April 1on1
- talked about travel from Sept. 12 - Oct 9th, missing first 2 weeks of Autumn ’23 quarter, focusing that quarter on writing & analysis
- GOAL: have all sequences (16S & Transcriptomes) by September 1st 2023
- Initial 20 samples extracted & sent for sequencing before FHL
- 2nd committee meeting to be held once initial 20 samples are run through 1st analysis for 16S & DGE, get advice on strategy for selecting remaining samples
- Follow-up samples extracted & sent for sequencing in July/August
- Send draft letter for NAH experiential learning fund to JPG on Monday April 24th
Initial test round 2 samples extracted!!!
3-CA1a
3-Ea
- Sampes selected from extr1 using a random number generator
- details can be found in the coral-DNA-RNA-lab-extractions repository
20-APR-2023
- downloaded fastq files for ‘fake data’ to continue script for bioinformatics workflow
- Received Mortexer! Whoo!
- Received cryo-gloves
- got 8lbs of dry-ice with NH
- re-organized cryo-vials to wax boxes, with all of the ‘a’ samples in 2 boxes labelled with green lab tape and ‘b’ samples in 2 boxes labelled with blue tape with NH
- setup lab bench for dry run dna/rna extraction with NH
- NH setup mortexer
- submitted 2-year extended warranty for mortexer s/n 22040448 via benchmark scientific in JPG’s name
- placed signage on lab 236 to lint roll and lab coat (because I forgot to and that was embarassing)
- submitted purchase for mortexer attachment head that will homogenize 12 1.5-2mL tubes at a time horizontal orientation (even better because closer to original lysis protocol for Zymo) Sadly, I should have looked for something like this earlier, but I saw it on the mortexer instruction manual when it was delivered and realized that was what I needed
19-APR-2023
- continued draft application for ONeill-Quinn
- continued editing extraction protocol
- expected delivery of mortexer by end of day
18-APR-2023
- Met with Nora, selected samples for processing using R script to randomize
- Setup workflow in Hyak
- added to extraction protocol
- waiting for mortexer delivery, expected by end of day, but FedEx package delivery delayed
- emailed asking for details about Neptune controller unit (it was returned after being sent away for RMA maintenance request, but nothing appears to have been done to it)
17-APR-2023
- added to extraction protocol
- read about remote computing, Hyak Mox
- enabled Hyak account
14-APR-2023
- attended Cat Fong chalk talk & SAFS Cafe
- gave tour to Edith
11-APR-2023
NCBI SRA links downloading RNAseq fastq files
Looking for ‘fake data’ for M. capitata
DNA, ITS2 to measure symbiont to host cell ratios for ‘Patterns of bleaching and recovery of Montipora capitata in Kaneohe Bay’ hosted at zenodo here https://zenodo.org/record/53231#.ZDWE0HbMKTQ -RNAseq resources from A. Huffmeyer
- https://ahuffmyer.github.io/ASH_Putnam_Lab_Notebook/E5-Deep-Dive-RNAseq-Count-Matrix-Analysis/
- https://github.com/AHuffmyer/EarlyLifeHistory_Energetics > Here is the spreadsheet of my NCBI accession numbers for my sequences: https://docs.google.com/spreadsheets/d/1qDGGpLFcmoO-fIFOPSUhPcxi4ErXIq2PkQbxvCCzI40/edit?pli=1#gid=457642286 If you look at the Mcapitata Ontogeny Timeseries tab it’ll have all the SRR numbers you will need to download the TagSeq files. Instead of downloading the full data set you could just pick like three lifestages for a test comparison - AH
10-APR-2023
- created script to randomize sample processing in coral-DNA-RNA-lab-extractions repo
- called Zymo and learned that ‘mix’ in protocol step can either mean pipette up and down 10-12 times, or briefly touch to vortex mixer for a pulse
- Zymo tech also shared that it’s best to follow protocol and add Lysis buffer to the sample supernatent in a separate rnase-free tube. If you do it in the filter tube some sample may start to percolate thru the filter before being fully buffered.
- Horizontal Microtube Holder
March 2023
29-MAR-2023
started template for DNA/RNA data extraction sheet in new repo for extractions
organized notes for samples in metadata
installed git on WSL via this tutorial
finished homework 1 for FISH546
lab-meeting notes
purchasing
no purchases in June!
The University of Washington is transitioning from their old DOS-based system to Workday Finance, with a Go Live date of July 5th, 2023. During the transition period, there will be some disruptions to normal operations, and the continuity of research and teaching and minimizing the impact on faculty, staff, and students is the priority. There will be Restrictive Periods during which no one will be able to process purchase orders or obtain reimbursements in ARIBA. Exceptions may be made for urgent or emergency purchases. Travel reimbursements and non-travel-related reimbursements should be submitted before June 9th, and processing will resume on July 5th. Blanket Purchase Orders that are expected to be active past June 30th, 2023, will not be automatically transferred to Workday, and all continuing BPOs must be manually reentered by the College of the Environment. Navigating the transition will require patience and close partnerships between research and administrative groups
space survey
- % of time spent in spaces from July ’22 - March ’23
- 2% 242
- 8% 236
- 30% HIMB
- 60% 264, 264B
- undergrad Nora Hessen 2hrs per week in 264, 264B since January ’23
28-MAR-2023
read papers:
called Zymo tech Christian Camacho ccamacho@zymoresearch.com to get advice on working with coral samples and the Zymo DNA/RNA miniprep kit:
bead bash for 40 minutes for microbes on mortexer vortex-mixer! This is still tested to result in good RNA, though there may be a trade-off between bead-bashing for lysis and RNA interity… see Zymo optimized lysis protocol
benchmark mortexer is not a tested method for the kit.. but it’s closest to the vortex genie approved method for lysis
mortar & pestle with liquid nitrogen, then add powder to bead bashing tube with RNA shield
lyophilized (freeze-dried) proteinase K is stable at room temp, it should be stored in the -20 freezer only after resuspending in the buffer
the DNAse 1 is similarly a lyophilized powder, but a very small amount, such that the vial looks empty
Gathered lab supplies
- mortar & pestles
- alcohol burner for use with denatured ethyl alcohol
- flint spark torch ignitor
- found expired RNA later
- DNAse away
- DEPC water (does that expire?)
- weigh papers (for powdered samples)
NEED
- scoopulas (to scoop powder from mortar to weigh paper)
- denatured ethyl alcohol (disinfect labware between samples)
27-MAR-2023
read papers:
- https://environmentalevidencejournal.biomedcentral.com/articles/10.1186/s13750-023-00298-y
worked on coral collection report
21-MAR-2023
- interviewed for WSG student assistant position
- began work on himb-2022 coral collection report
20-MAR-2023
- installed Sophos anti-virus on Owl
- attended Robert’s Lab meeting
- checked out Qubit buffer + reagents in Roberts lab with Olivia & Zach
- put mortexer, DEPC-treated water, Qubit assay kit, on purchase log
- wrote Qubit RNA Broad Range Protocol post
16-MAR-2023
- finished SMEA/FISH 539 final paper
15-MAR-2023
- out sick
- worked on SMEA/FISH 539 final paper
14-MAR-2023
- out sick
- talked with Hailey Dockery about shadowing RNA extractions
13-MAR-2023
- worked on SMEA/FISH 539 final paper
- updated R & RStudio to latest versions
10-MAR-2023
- finished SMEA/FISH 1page reflection
- worked on SMEA/FISH 539 final paper
- met with GSIS speaker on Zoom
- made hotel reservations for GSIS
09-MAR-2023
Attended Coastal Ecology candidate chalk talk
Shadowed Grace for new RNA extraction protocol & Qubit QC for RNA quality
Met with Emma Strand about M cap DNA/RNA extractions… notes from Zoom chat:
- email Erin Chille eec72@scarletmail.rutgers.edu about 16S extraction kit
- https://emmastrand.github.io/EmmaStrand_Notebook/Kbay-Bleaching-2019-DNA-RNA-Extractions/
- host DNA (looked at DNA methylation), symbiont DNA (ITS2), microbiome DNA (16S not so good), host RNA + symbiont RNA (Illumina Nova Seq RNAseq)
- coral issues- mucus clogging spin-column filter (avoid mucucs, avoid skeleton)
- how big of a fragment do I put in the bead tube? 1/3 bead, 1/3 coral, 1/3 shield – https://emmastrand.github.io/EmmaStrand_Notebook/Kbay-Bleaching-2019-DNA-RNA-Extractions/
- optimize time to vortex bead tube (2mins for m. cap) *vortex machine, holds 8 tubes at once
- https://www.southernlabware.com/mortexertm-vortex-mixer-115v.html?gclid=Cj0KCQiApKagBhC1ARIsAFc7Mc5LH-QjqtDR98g9ZgE9iAOs-y35m7X5rr7pHCqfPGRtTJVPEeQWbLYaAtCpEALw_wcB
- 100uL total DNA material end product from kit
- 12ng of input for ITS2 about 5uL
- aim for 10 - 70 ng/uL DNA quantity (Qubit)
- DNA quality (gel) *important for methylation studies, but may not be for 16S/ITS2
- 100uL total RNA material end product, send 20-30uL for RNAseq
- https://github.com/hputnam/HI_Bleaching_Timeseries/blob/main/Dec-July-2019-analysis/data/Molecular-labwork.xlsx
- sample ID, use a separate extraction ID (in the event of multiple extractions from the same sample!)
07-MAR-2023
- worked on SMEA/FISH 539 final paper
- met to make GSIS budget w/ Juliette
- met with Grace from Robert’s Lab to practice RNA extraction
- participated in SAC interview for ADDEI
06-MAR-2023
- presented final group project in SMEA/FISH 539
- worked on SMEA/FISH 539 final paper
- attended Coastal Ecology faculty candidate seminar
03-MAR-2023
- I swear I’ve done stuff since January / how has winter quarter gone by so fast?!
- submitted PCC Climate Solutions grant funding application this morning
- worked on FSMEA/FISH 539 final project + presentation
02-MAR-2023
- submitted FHL TA application for summer A term marine botany
- helped interview ADDEI candidate
- attended Carter’s Coastal Ecology candidate chalk talk
- met with Steven on Zoom and got permission to use Bioanalyzer
- worked on sequencing quotes, DNA/RNA extraction protocol, reached out to Emma Strand and Grace
January 2023
31-JAN-2023
- prepared 1st committee meeting blog post
20-JAN-2023
- cleaned PBB
- strategized chapter 1: Low Dose Effects & chapter 2: Multiple Stressors for thesis
- arranged to meet with Miranda to talk about summer kelp gametophyte project
- completed lab safety trainings
19-JAN-2023
read Ariana Huffmyer’s lab posts on extraction protocols
read Ainsworth et al 2015 & Brown et al 2021
- MarBio Plant DNA extraction Kit for whole coral holobiont 16S rRNA Bacteria
- Pure Link Plant Total DNA purification systems (Invitrogen) for 16S rRNA Bacteria
attended JPG lab meeting and gave feedback on Eileen Bates’ pinto abalone presentation for Alaska Science Sympsosium
designed graphical methods in Canva for pae-temp experiment
18-JAN-2023
- worked with norahess to setup collaboration on ibis repo!
- talking about setting up Aiptasia spawning tanks to run experiments with cnidarian model organism reproduction
17-JAN-2023
- attended Roberts lab meeting, discussed curriculum development for Maritime Highschool
13-JAN-2023
- Read up on ‘Omics’, started a post on definitions and basics of the study of DNA & RNA
- Found whole genome for M. capitata on NCBI
- Helmkampf, M., Bellinger, M. R., Geib, S. M., Sim, S. B., & Takabayashi, M. (2019). Draft Genome of the Rice Coral Montipora capitata Obtained from Linked-Read Sequencing. Genome Biology and Evolution, 11(7), 2045–2054. https://doi.org/10.1093/gbe/evz135
- Williams, A., Pathmanathan, J. S., Stephens, T. G., Su, X., Chiles, E. N., Conetta, D., Putnam, H. M., & Bhattacharya, D. (2021). Multi-omic characterization of the thermal stress phenome in the stony coral Montipora capitata. PeerJ, 9, e12335. https://doi.org/10.7717/peerj.12335
- Shumaker, A., Putnam, H. M., Qiu, H., Price, D. C., Zelzion, E., Harel, A., Wagner, N. E., Gates, R. D., Yoon, H. S., & Bhattacharya, D. (2019). Genome analysis of the rice coral Montipora capitata. Scientific Reports, 9(1), 2571. https://doi.org/10.1038/s41598-019-39274-3
12-JAN-2023
Explored the use of purple sea urchin and/or Aiptasia for phthalate low-dose effect gametogenesis study (proposal brainstorming) for the IBIS research mentor program
Created ibis repo to organize project
Completed lab safety training matrix for myself and NH, identified required and recommended lab safety trainings
Potential to use rack in PBB near interior door for Aiptasia spawning incubator, setup workflow with dissecting scope and camera all in PBB for IBIS project
Inventoried equipment we have in lab 236 for molecular work:
- Beckman Coulter Allegra X-30R Centrifuge
- Thermo Scientific NanoDrop 2000c Spectrophotometer
- Fisherbrand Isotemp heat block
- Beckman Coulter Microfuge 16 Centrifuge
- Eppendorf Mastercycler nexus gradient
09-JAN-2023
Today I setup my GitHub ‘notebook’! This is the beginning of my exploration of using GitHub to jot down thoughts, record ‘daily bits’, and organize the procedures and methods I’ll use in my thesis research
zbengt introduced me to ‘elicit’, a lit review search AI. I searched “What is the role of endocrine signaling in cnidarian development” and found an exciting paper titled “Sea Anemones Responding to Sex Hormones, Oxybenzone, and Benzyl Butyl Phthalate: Transcriptional Profiling and in Silico Modelling Provide Clues to Decipher Endocrine Disruption in Cnidarians” authored by Morgan et al. in 2022
I explored another AI, ChatGPT, and gave it the prompt “write me a poem about corals and oysters and mothers” and it returned:
Coral reefs, a vibrant sea of life, A home for countless creatures, rife. Oysters, clams, and mussels too, All nestled safe, their shells in view.
But danger lurks, the waters warm, The coral bleaches, it’s colors torn. Mother Nature’s wrath, a sight to see, But we can help, if only we believe.
For mothers, fierce and loving beasts, Will do what it takes, to protect at least. Their young and home, with all their might, They’ll fight and strive, through day and night.
So let us join, in their noble quest, To save the reefs, and all the rest. For oysters, corals, and mothers too, Deserve a chance, to make it through.
I followed up with a ChatGPT prompt “what are examples of endocrine signaling in cnidarians?” and it returned:
Endocrine signaling is the process of communicating information from one cell to another by releasing hormones into the circulatory system or through direct contact. In cnidarians, endocrine signaling can be used for a variety of functions, including the regulation of growth, development, and behavior.
One example of endocrine signaling in cnidarians is the release of growth hormones, which stimulate the growth and development of cells and tissues. For example, in some species of cnidarians, the release of growth hormones is regulated by the action of a hormone called insulin-like growth factor (IGF).
Another example of endocrine signaling in cnidarians is the regulation of reproductive function. In some species of cnidarians, hormones called gonadotropins are released by the endocrine cells of the gonads (sex organs) to stimulate the production and release of gametes (sex cells).
Endocrine signaling is also involved in the regulation of behavior in cnidarians. For example, some species of cnidarians release hormones called neuropeptides that act on the nervous system to regulate behaviors such as feeding, movement, and response to stimuli.
Overall, endocrine signaling plays a vital role in the regulation of various functions in cnidarians, including growth, development, and behavior.
**Is this is all true!? I’ll have to follow-up with my own dive into primary literature…
Another ChatGPT AI response, when asked the same questions, but prompted to provide references:
Examples of endocrine signaling in cnidarians include the release of hormones in response to stimuli such as touch, temperature changes, and changes in light intensity.
For example, the hormone adenylate cyclase-activating polypeptide (ACAP) is released from cnidocytes in response to a variety of stimuli and activates adenylate cyclase, leading to the production of cyclic adenosine monophosphate (cAMP) and the regulation of various physiological processes.
Other signaling molecules that have been identified in cnidarians include cyclic guanosine monophosphate (cGMP), inositol triphosphate (IP3), and Ca2+-dependent signaling pathways.
References:
Levitan, D. R., & Kohn, A. B. (2001). The Endocrine System of Cnidarians. Annual Review of Physiology, 63(1), 319-344.
Schmid, V., & Pfründner, R. (1995). Adenylate cyclase-activating polypeptide in cnidarians: a new class of peptides with signal transducing properties. Regulatory Peptides, 61(2), 149-157.