Goals for August
SAFS Work
- Receive Edits from Proposal and Submit
Goals for July
SAFS Work
- send Proposal to comittee members for approval
Goals for June
SAFS Work
SAFS fellowship- CICOES 2022-23 Graduate Student Fellowship (June 30)
- Write proposal and send to comittee members for approval
Goals for April
Experimental Work
- Rerun any missing data from CS run on 3.21-3.22
Circos Plot Tutorial (on Raven)
Tutorial for Circos plots
Functional Annotation of P. Generosa transcriptome part 6
Updated Counts and Overlapp
Total Set Size
Goals for March
Experimental Work
- buy pipette tips to finish Matt George’s CS activity experiment
- buy camera for flourescent scope
- order 1 more CS kit
- keep testing flourescent scope
- optimize CS protocol for reducing pipette tips
- Run BSA and CS on 72 of Matt’s samples
Functional Annotation of P. Generosa transcriptome part 5
Note: I was using the wrong UpSet plot data format:: I redit this entire pipeline in the post from 03-11-22
Functional Annotation of P. Generosa transcriptome part 4
Following steps after Functional Annotation of P. Generosa transcriptome part 3
overlapping genes counts by tissue type
Functional Annotation of P. Generosa transcriptome part 3
First Step: take merged data from geoduck transcriptomes and upload to R
countMatrix<-read.table(file="https://raw.githubusercontent.com/sr320/nb-2022/main/P_generosa/analyses/kallisto-0207.isoform.counts.matrix", header=TRUE, sep = '\t')
names(countMatrix)[1]<-"target_id"
head(countMatrix)
Second Step: take blast data and load
#load blast data, see blasting .sh file to see code (in GItHub)
blast_data<-read.table(file="https://gannet.fish.washington.edu/gigas/Panopea-generosa-uniprot_blastx.tab")
names(blast_data)[1]<-"target_id" #renamed to match countMATRIX data
names(blast_data)[2]<-"identifiers" #renamed to search in uniprot swiss prot
Third Step: merge blast data with geoduck transcriptome data
Goals for February
experimental work
Goals for January
experimental work
Functional Annotation of P. Generosa transcriptome part 2
Final Link to cleaned count data with Go terms pgenerosa larval gene annotations
Goals for December
experimental work
BSA script
Read MoreCS script
Get Standardized protein concentration (ug/mL)
protein_test<-read.csv(file="/Users/oliviacattau/Documents/data/BSA_Nov22_mantle+gill.csv")
A<-protein_test$Absorbance...570..1.0s...A.
protein<-(A*2500)-1700 #from GLM standard equation
protein_test<-cbind(protein_test, protein)
protein_test<-protein_test[protein_test$protein >= 0,]
M1= subset(protein_test, protein_test$label=='M_1')
M2= subset(protein_test, protein_test$label=='M_2')
G1= subset(protein_test, protein_test$label=='G_1')
G2= subset(protein_test, protein_test$label=='G_2')
M_1<-mean(M1$protein)
M_2<-mean(M2$protein)
G_1<-mean(G1$protein)
G_2<-mean(G2$protein)
protein_list<-c(M_1, M_2, G_1, G_2)
tissue_label<-c("mantle_1", "mantle_2", "gill_1", "gill_2")
protein_content<-as.table(cbind(tissue_label, protein_list))
print(protein_content)
CS activity (nmol/min/uL)
CS_Nov22<-read.csv(file="/Users/oliviacattau/Documents/data/CS_Nov22.csv")
CS_Nov22<-CS_Nov22[!(CS_Nov22$Well=="B06" | CS_Nov22$Well=="B03"),]
A<-CS_Nov22$Absorbance...405..0.1s...A.
blank=subset(CS_Nov22, CS_Nov22$label==0)
zero<-mean(blank$A)
new_A<-A-0.0664 #subtract blank well
CS_Nov22<-cbind(CS_Nov22, new_A)
M1= subset(CS_Nov22, CS_Nov22$label=='m1')
M2= subset(CS_Nov22, CS_Nov22$label=='m2')
G1= subset(CS_Nov22, CS_Nov22$label=='g1')
G2= subset(CS_Nov22, CS_Nov22$label=='g2')
CS_tissue<-rbind(M1, M2, G1, G2)
#plot data
library(ggplot2)
GSH<-ggplot(data=CS_tissue)+geom_point(aes(x=as.factor(Time), y=new_A, color=label))+theme_bw()
GSH
time_0<-subset(CS_tissue,CS_tissue$Repeat==1)
time_30<-subset(CS_tissue, CS_tissue$Repeat==30)
time_0$delta<-time_30$new_A-time_0$new_A #delta OD to get GSH
standard_CS<-read.csv(file="/Users/oliviacattau/Documents/data/standard_curve_small.csv")
cs_model<-lm(standard_CS$New_A~(standard_CS$GSH.well))
summary(cs_model)
cs_equation<-paste("y=",coef(cs_model)[[1]], "+", coef(cs_model)[[2]], "*x")
cs_equation
time_0$nmol<-0.0753742256190483 + 0.0465124174107143*delta #equation from GSH standard curve
standard_cs_plot<-ggplot(data=standard_CS,(aes(x=New_A, y=GSH.well)))+geom_point()+geom_smooth(method="lm", se=FALSE, formula = y ~x)+xlab("OD")+ylab("GSH")+theme_bw()+stat_regline_equation(label.x=1, label.y=8)
standard_cs_plot
#get CS activity
B<-21*delta-0.73
V<-50 #uL
D<-1
time<-27 #min
SampleCSactivity<-B/(time*V)*D
time_0$CSactivity<-SampleCSactivity #(nmol/min/uL)
|tissue_label| OD (delta A)| GSH nmol| CSactivity (nmol/min/uL)| protein (ug/mL)| protein (ug/uL)| relative CS activity (nmol/min/ug)| |—|—|—|—|—|—|—| |mantle_1| 0.116| 1.706| 1.26E-03| 493.89| 0.493888889| 2.56E-03| |mantle_2| 0.527| 10.337| 7.66E-03| 89.17| 0.089166667| 8.59E-02| |gill_1| 0.216| 3.806| 2.82E-03| below range| NA| NA| |gill_2| 0.08| 0.95| 7.04E-04| 445.83| 0.445833333| 1.58E-03|
BSA check
bovine serum albumin (BSA) test
Goals for November
experimental work
Functional Annotation of P. Generosa transcriptome
Inspect P. generosa genome for candidate genes. First step is to extract output data from kallisto:
steps on how to create kalllisto index file
Read MoreLab work Mondays
Check to see if BioRad bovine serum albumin (BSA) Protocol works with oyster tissue
if it does, use the results from the assay run to calculate total protein concentration per sample (ug/mL)
I am going to want to use various dillutions of the samples so I can get the protein concentration in the linear range (<2000 ug/mL)
Goals for October
experimental work
Citrate Synthase technical replicates
Citrate-Synthase (CS) Test Run with Technical Replicates
Citrate Synthase test 2
CS Test #2
Ran the sample curve, background control and positive control for the CS kit on the spec in FTR. At 405um instead of 412um, the sample curve was uneffected by the slight change in wavelength. Monday I will run another test with oyster tissue to test the enzyme at 25C with the old machine.
Citrate Synthase test
Citrate-Synthase (CS) Test Run #1 Today Matt George and I attempted to test out our CS protocol using the Varioscan spectrophotometer. The original settings on the machine was 1000 meaurements without any breaks, but at 25 seconds a measurement, that would mean the Variscan would have been running for 12.5 hours so I had to kill the scripts which deleted the data. I re-ran the machine 57 minutes later (20 minutes past the ideal time) with the new settings (30 measurements with 2 second breaks=12.5 minutes of samples) and captured the very end of the absorbance curve with the absorbance spectrum centered at 412um. Results from the first test run can be found here
Goals for Septemeber
Goals for September
Kallisto Index for P. generosa
using Roadrunner for bulding a Kallisto index
updates
The worst happened yesterday,
my precious Prius way violated at Safeway and they took my baby! (By baby I mean my Macbook air that I had since college) and my smartphone. For the last 48 hours I have been without a phone and I realized how I rely on my phone to authenticate my identity. I cannot log into my apple ID without a 2-factor authorization which is problem because without my phone I cannot restore my new laptop to it’s time machine back-up. Thankfully, I have an external hard drive that has all my college work on it but I didn’t back it up since I started at UW this spring. All my work is on GitHub but my computer is empty which means that I am starting over from scratch with Jupyter Notebook. Maybe it will be a good exercise going through installing homebrew and kallisto again, so I know how it works intuatively. I have been stuggling with understanding where the computer stores my files so this should be a good opportunity to “clean up” my work.
Goals for August
August 2021 goals
-finish procedure for Citrate-Synthase Assay, start testing samples when the kit arrives -plan for next experiment with Steven on oyster breeding -catch up on all coding homework -watch the rest of the OLYMPICS for inspiration -think about TA positions for next school year -think about what classes I want to register for in the Autumn
Citrate-Synthase Assay
Today I wrote out the procedure for the citrate-synthase assay link to google doc here
Last Day at Pt Whitney
End of an ERA
Today was the last day (Monday July 26th 2021) at Point Whitney working with Matt George (mngeorge@uw.edu) on oyster disections for his pacific oyster triploid temperature stress experiment.
death becomes geoducks
Updates on Geoduck Project
On July 15th I went to the hatchery at Pt Whitney to check mortality for Matt George’s maturation control project. While I was there I also checked on the seasonal fertility of my geoducks -Results: I re-labeled the remaining 6 animals with with F or M (circled) to indicade Male or Female, and then I assigned them a letter A,B,C,D,E,F (underlined). In the case of animal F, it was a male so I gave it a number #6. -Fertility: I pulled a sample of the gonad with a syringe (non-fatally) and there was no ripe females and only a few males with active sperm. It is not the spawning season right now and these animals might have to overwinter at the hatchery to re-ripen for buoyancy experiment.
July goals
Goals for July research 2021
First Project: lab work 2-3 days a week on the Pacific Oyster triploid temperature stress experiment #2 with Matt at Pt Whitney -So far I have been labeling oysters, measuring shell lengths, and feeding rate assays. Will eventually be in change of the citrate synthase assay as well.
learning how to characterize larval/juvenile geoduck transccriptomes
2021 May 12th
library(credentials) set_github_pat()