Today I worked more on my R script for adding new Qubit files. Everything works great up until the actual joining of files. After joining, there are extra columns that have the extensions “.x” and “.y”… I think it has something to do with the fact that some columns are factors, some are characters, and some are numeric… I also started using Mox today, but am unsure how to upload the .fastq files from the C bairdi transcriptome data. Waiting to hear back on that in a GitHub issue.
Today, Steven, Sam, and I discussed where we’re at with the crab samples and RNA extraction. We decided to extract some samples using the RNeasy Mini Plus Kit from Qiagen that Sam has already used and had decent results. In the meantime, I’ll be working on creating a little metadata analysis comparing the different methods used so far, the yields, whether the samples were lyophilized, etc. To do this, I had to figure out how to add the new Qubit data to the crab sampling file. I think I’ve figured out a general work flow in R, and Sam helped me out with some codes to fix some errors I was having.
Today I extracted RNA from 10 more pelleted hemolymph samples using Tri-Reagent (without lyophilizing). I ran 5 ul on the RNA HS Qubit, and 7/10 had quantifiable RNA. I also started working with the .fastq sequence data from our first sequenced library. I ran the four files through FASTQC and am learning how to determine if the output is good.
Today I extracted RNA from 10 samples from Day 26. These still had all three samples in the box, so they are all ###-1 tubes. The QUbit wouldn’t let me download the data from the USB. I’m not sure why, but luckily I wrote down, as I always do, the reads for each tube number. Out of the 10, 7 had quantifiable RNA by the RNA HS Qubit.
Today I discussed with Sam about how to use R for making a better master file. We identified some short term and long term goals for me to work on (and get help with via GitHub Issues). I also ran the Bioanalyzer on Test3 (from when lyophilizer was used and using Tri-reagent ), and four samples that I extracted last Friday using (wihtout lyophilizer, with Tri-reagent). The Bioanalyzer didn’t run great because the ladder and the markers didn’t show up… will have to re-run.
Today I extracted RNA from four practice samples using Tri-reagent, and without lyophilizing (lyophilizer is out of commission again for now).
- Identify successful RNA extraction protocol
- Get extracting RNA going so that we can plan to send some more samples for library prep and sequencing
- Publish more DecaPod episodes (about 2 - one every two weeks)
Today I submitted DecaPod RSS feed to Apple for approval to be published on iTunes! I also picked out some tubes for Tri-reagent RNA extraction without lyophilizing (it’s broken again) for Monday or Tuesday next week to get things moving again.
At 10:30 am, I went to the lyoppilizer room and met up with Megan. My samples had gotten all over the inside of the manifold. Sam thinks it’s likely because the sampels thawed before they got on the lyophilizer, which is very likely because I brought them over on wet ice as opposed to dry ice. WIll try again at 4:30pm (bringing them over on dry ice) and Sam will grab them in the morning on Thursday. I will also test this out only with screw cap tubes and have them slightly unscrewed. Megan also noticed that the vacuum didn’t get below 100 mtorr, which it should… so the manifold likely wasn’t sealed properly. So Megan added a lot more grease.
Today I started using the lyophilizer on the samples I picked out (pellet and supernatant) and the pooled sample of the ones from Sam’s Qiagen RNeasy extraction that had “Out of range” Qubit results.
Today I edited and published DecaPod S1E11: Crab Meeting #5. I also have a plan for the format of the podcast.
Today we had Crab Meeting #5. We discussed the new plan for using the lyophilizer (freeze drier) and Tri-Reagent protocol for extracting RNA. I recorded the meeting and will edit and publish tomorrow as a new DecaPod episode. I also went over to FSH building with Sam and he showed me how the lyophilizer works. On Monday, I will put the samples in and retrieve them on Tuesday, at which point they will be ready for Sam to extract RNA from and for me to run the lyophilized pooled sample on the Qubit.
2015 Oysterseed Project:
- Figure out Skyline DIA with Emma (plan for 9/6/18)
- Analyze 2015 Oysterseed data
Today I re-tried the Skyline DIA 2015 Oysterseed protocol. Emma has been pretty busy teaching her course these past several weeks and hasn’t been able to get back to me on my GitHub issue (#341). I re-did the protocol using the same files and quadruple checking all the settings. Things still look mediocre. I’ll post the new .zip to today’s file on the GitHub issue so she can take a look at it when she has time.
I’ve been sick the past several days, and I’m finally feeling well enough to come in to SAFS. Today, since I’m still not back to normal, I spent the day reading some background information on SRM, Targeted proteomics, and general crab biology. I also outline some goals I have from now until the start of school on September 25th.
Today I finished speed vac-ing (medium heat) the pooled sample. It ended up being too low of volume (14.1ul), so I added 40.9ul of 0.1% DEPC-treated H20. I ran 2ul of the sample on Qubit (RNA HS), and got a reading of 20.4 ul (1,081 ng RNA in the sample)!!!! We FINALLY have a sample to send off for library prep and sequencing! After getting info from NWGC (Chris in the Nickerson Lab at Foege) I put the sample on dry ice and walked it over! It is now in their hands until we get the data back. :)
Today I speed vac-ed the new pool of 15 samples for a little over three hours. There is still more than 50ul in the tube, so I’ll put it back in the speed vac as soon as I am in tomorrow morning.
Today Sam, Steven, and I met and decided to pool the 15 samples that had quantifiable RNA (Qubit RNA HS results) from the 40 samples that Sam processed using the Qiagen RNEasy Mini Plus Kit into one sample to be sequenced. Also, they both helped me learn more about data management and creating a better file that contains the crab hemolymph sampling data and the Qubit results data.
Today I played around with GitHub and R Projects. I’ve figured out a way of making adding Qubit results to my hemolymph and Qubit results master file more easy. It isn’t perfect and there are likely some things that I don’t know about that could make it easier, but I’ve detailed the new method below. I also noticed today that the tube_number column in what I did yesterday got all messed up with the merge in R. Detailed below.
Yesterday I shared some preliminary thoughts (in a GitHub issue and detailed below) on what we can do for our first Crab RNA-Seq library. Today I learned - through a whole mess of comments on a GitHub Issue because my stuff isn’t organized well - how to merge data in columns that are the same in R, so that you don’t have duplicates (example: “Test_Date.x” and “Test_Date.y”). Steven mentioned I should go over with him how to name files… which sounds good to me because I think I’m taking too much time out of my day renaming and replacing files as I update them. Sam also mentioned that my R Project is difficult to work with because it includes files that belong to two different repositories. So I’ll work on cleaning that up and making it easier for future collaboration and help.
Today I met with Steven and Sam to make a new plan for the crab RNA problem. We’re going to use the lyophilizer (freeze drying machine that goes below -80˚C) on 8 new pelleted hemolymph samples from Day 26; 8 supernatant samples that correspond to those 8 pelleted; and a pooled sample of all the ones Sam processed using the Qiagen RNeasy Kit that had “out of range” Qubit results. This will have to be post-poned for a bit becuase the lyophilizer is currently being fixed… waiting for an ETA on that. In regards to Skyline, I re-visited what I did before I left for vacation and went through all the files used and settings chosen. I put all of the information in a new GitHub issue per Steven’s request.
Today I noticed that the code Yaamini showed me yesterday to change the “tube_number” column values in the 20180522-all-crabs-hemo.csv from Factor to numeric ended up changing the tube numbers, which meant that the spreadsheet I made yesterday was wrong. So, Sam taught me how to make the “tube_number” column from Factor to numerics without changing the value, as described below.
I am picking some samples for Sam to use an RNeasy Mini Plus Kit on. They are from Day 26 (samples were taken in triplicate), and 10 were picked from each of the following groups: uninfected, cold; infected, cold; uninfected, ambient; infected, ambient. I also made a spreadsheet that contains the hemolymph sampling data of all the crabs in the whole experiment (including those that died) left_join-ed with Qubit results data. However, after going through my Qubit csv folder, I found a couple of .csv’s that didn’t make it into the consolidated qubit csv. So I am currently trying to join the hemolymph data csv with the UPDATED Qubit results data based on tube_number, but am getting an error.
- Continue working with Sam and Steven to find solutions for the RNA isolation issues I had
- Finish creating a good, readable spreadsheet of all the crab data
Today I ran the Bioanalyzer on the samples Sam isolated RNA from months ago using RNAzol RT as well as some of the RNA isolated from samples that I have done. The results were not good - no dye showed up, so maybe I did it wrong. TBD. Also, Sam used a kit to clean up the RNA that I have isolated and then ran Qubit and Nanodrop1000, as well as used RNeasy Plus Mini Kit to isolate RNA from untouched hemolymph samples. Low concentrations of RNA, but at least there is RNA. The results from the RNeasy Plus Mini Kit isolations are clean and the RNA on Qubit and Nanodrop1000 are good.
Today Steven, Sam, and I met to talk about the issue with poor results on the QUbit and Nanodrop1000 on the pooled hemolymph samples I prepared. Short-term plan is to try out the RNA protocol, but multiply all reagents times 6 (so, use 6ml of RNAzol, etc.) to see if that ratio would help; run the Bioanalyzer on the samples that Sam processed as well as some random samples that I isolated. Today, I did the 6x RNA protocol on 4 of the extra samples from day 26 (samples from day 26 were taken in triplicate).
Today Walnut finished up using Walnut to make a new BLIB file with the 2015 C.gigas Oysterseed raw files. Then, I used the new BLIB in SKyline, along with changing the minutes to 5, and double-checking I used the same .mzML files used in Walnut as my results in Skyline. The peaks still look pretty bad. I just emailed Emma…
Today I started using Walnut (upgraded PECAN) to create a new BLIB file for the 2015 Oysterseed project. Hopefully this will improve the error rates in Skyline. Also, I called Pam to work on the NPRB progress report. Additionally, I detail Sam’s updates on the status of the Crab pool samples from his notebook post. The RNA needs some cleaning so he suggests trying RNeasy Cleanup Kit. I will wait until he returns Monday and speak in person with him and Steven to figure out what to do next.
Today Sam speed vac-ed the pools and put them on ice. We mixed the samples and ran Qubit. The readings were way too low. Sam got out hte Nanodrop and we ran them on that… and the readings were bad. Since I am out of town the rest of this week and half of next, Sam is going to try to fix some things, detailed below. In terms of Skyline, Emma has been in contact with Nick today and others from the Skyline crew. She is working on figuring out what the issue is with my super high error rates. At the end of today’s post, I also outline rest of July goals and summer goals.
CRAB RNA POOLS: Today I started using the Speed Vac on the three pools with Sam. They were running on low temperature from 10:30am to 1:15pm. Not much liquid had evaporated. From 1:15pm-3:15pm they were run on medium temperature. Still not enough liquid has evaporated, so Sam will put them in the Speed Vac when he gets to FTR tomorrow morning. SKYLINE: I tested out the tutorial that the people from Skyline suggested I try out. Not sure how it turned out honestly, but Emma said she can take a look at it with me tomorrow.
This post is a summation of what I did this week. I made the 3 pools for RNASeq (speed vac will happen Tuesday when both Sam and I are in), and am starting to create an ultimate master crab spreadsheet with ALL the data that we have on this project.
Today we had our 4th crab meeting and discussed our short-term sequencing plan for 3 libraries (1: day 9 uninfected; 2: day 9 infected; and 3:a “masterpool” from the reamining 10 treatments). We also discussed our plan going forward with qPCR and creating libraries later and we hopefully will see some cool things with the three current chosen libraries and the qPCR.
Today I met with Sam and Steven a couple times to figure out what we’re going to discuss this Thursday with Pam. Steven and Sam have come up with three pools that we feel are a good place to start because unfortunately there’s not enough RNA to do the original pooling plan. This post details all the info that I currently have on the plan for our discussion on Thursday.
Today I did more reading on how to start assembling a transcriptome using Trinity with plans to formulate the beginning steps tomorrow and I interviewed Genevieve Johnson (University of Alaska, Fairbanks) on her thesis project doing Tanner Crab population genetics for a new soon-to-be published episode of DecaPod.
Today I went through all my setting and files in Skyline for the 2015Oysterseed proteomic project and re-did my Skyline Daily peak-picking error rate determination. I still got an error rate nearing 50%, so per Emma’s suggestion, I submitted the issue to Skyline’s support page.
Today I met with Steven to talk briefly about the crab sample pooling (need more info on how to proceed- will wait for Sam’s return in 1.5 weeks), and about things I can do in the interim while the samples are being sequenced: practicing Trinity with Geoduck transcriptome data; DecaPod; 2015 Oysterseed project.
Today I worked on trying to figure out what could have made my error rates from last week so awful. I re-did the Skyline DIA protocol many times and ended up realizing that some of my samples only had a few files listed in the protein, while others had all four. Asked Yaamini for clarification. I was told by Emma to make sure that I have four tabs in Skyline. Each tab represents a sample, and each sample has 4 files associated with it.
Today I re-did the Skyline peak-picking error rate for the 2015 Oysterseed DIA and sent off the results to Emma for feedback. I also worked on figuring out how to make the 12 pooled samples that will be sent off for sequencing. Awaiting Steven’s feedback on that.
Tips and tricks for Jekyll notebook posts.
Today I picked samples for sending off to get sequenced. Obviously I have to get this checked with everyone… and I’m guessing they’d rather see it in a spreadhseet form and not color-coded as I do now… have to figure out how to do this.
- DIA error rates and move forward
- Pubathon for 2015 Oysterseed paper
Isolate RNA from warm crabs day 9 and 12 (crabs didn’t make it to day 26). Work more on creating a master file of all data associated with the crab project.
Plan for what I want to get done this week.
Trying out a join in R to figure out what I have in terms of isolated RNA samples.
Images from my trying out the bioanalyzer. And I try again. Neither tries look great. I should try again. Pub-a-thon plan and DecaPod S1E7 published (crab mtg #3).
- Create a visual representation of all of the successfully isolated RNA samples I have and figure out a pooling scheme for next Crab mtg
- Publish new podcast episode every week
- Run bioanalyzer on some samples
- Work on google doc for project
- READ MORE LITERATURE ON CRABS, HEMATODINIUM, TRANSCRIPTOMICS AND OTHER “-OMICS”
Today we had Crab meeting #3 and I tried out the bioanalyzer.
Isolated MORE RNA, Yaamini helps me with organization in R. Plan for tomorrow.
More RNA isolations and plan for data sheet organization for the week. Plan to try out Bioanalyzer.
RNA isolation over weekend to replace those that had “out of range”. Edited and published S1E6 of DecaPod.
More RNA isolation to replace those that had “out of range” RNA qubit results. Ended up contaminating them all, so unusable. Starting my journey of using R for data organization.
Today Pam showed me what she does in excel to move info from one spreadsheet into another based on a shared column. Plan for picking crabs to replace those that had “Out of range” qubit results (either no RNA in sample I pulled out; no hemolymph sampled in first place; or so little that it’s below limit of detection.
Last batch of RNA isolation based on what I picked previously. Working on organizing data sheets to figure out exactly what I have. Plan for this week.
Updates on progress of RNA isolation, DecaPod, and plan for the next week.
So last week was unfortunately not as productive as I had planned as a result of some dog-sitting shenanigans. So, to help myself get back on top of things, I’m making a list of what I want to and will accomplish this week. Firstly, tomorrow I have a presentation in my FISH 511 class, so I will be focusing on that today and tomorrow. The rest of the week will be as follows:
Updates on RNA isolation and Qubit progress and DecaPod progress and plan. Finally post S1E1 on project background!
Result from Qubit of RNA isolation samples and new episode of DecaPod published! Episode on RNA isolation - S1E4.
Updates on RNA isolation progress, DecaPod progress, and Data management plan.
- RNA Isolation
- Isolate RNA from all crabs within the subset I selected (2 more sets of 9 to go)
- Run Qubit on ALL of the isolated RNA samples from the subset I selected
- Edit the three recordings I have that are about:
- RNA isolation
- Crab mtg #2
- Background info on the project (Ep. 1)
- Come out with an episode each week
- Edit the three recordings I have that are about:
- Organize into master spreadsheet - or at least start the process (morph data; qubit; EVERYTHING)
- Add Qubit results to the spreadsheet containing the subset of samples that I’m working with to isolate RNA
More RNA isolation - successful! Plan for tomorrow - Crab mtg #2 and work on DecaPod with Pam.
Today I continue RNA isolation using the newer version of the protocol. Successful day!
Today I use the updated version of the RNA isolation protocol and find that it is so much better and isolating RNA than the generalized protocol. This one is based on what Sam did with extra samples from Sam in the fall of 2017. It adds some extra mixing and centrifuging steps that appear to be the key. Good results.
Today I updated the RNA isolation protocol based on info from Sam, and I made some mistakes in isolating RNA today. Ran Qubit.
Today I finish the error rate determination in Skyline and hope that I did it right so that I can move on with the DIA analysis. I also run Qubit to quanitify the RNA in my samples I extracted from.
I isolate RNA from another batch of 9 samples (3 crabs) and I try to understand what I’m doing in terms of determining peak-picking error rate.
I re-do the DIA analysis from step one. I’m still unsure how to do error rate determination. The protocol is not clear on how to do this.
Get some input from Emma on DIA and I start isolating RNA.
Updates on RNA isolation sample picking; DecaPod; and DIA analysis progress.
I provide updates on status and next steps for my different projects.
My goals for this month are to continue my current work. I also look back on how I did in February.
Today I worked on organization the hemolymph crab data and went to a workshop hosted by UW IT to learn about Audacity for Podcasts.
Today we had our first crab project meeting. Our plan is to meet the first Thursday of the month (subject to change if need be).
Today I began the process of isolating RNA from the “official” crab hemolymph samples, although they can change if I mess things up, because there are many extra samples.
I figured out how to upload results in Skyline such that there are 4 tabs (because I have 4 samples) and each tab has four files associated with it. Moving foward in DIA (confusion in determining error rate, so I’m stuck); plans for tomorrow and RNA isolation.
Revisiting what I did last week and my goals for next week: DIA, podcast, RNA isolation.
This quarter I am taking FISH 521, which is the Research Proposal Writing and Professional Development course taught by Julian Olden. This class has been very helpful because it has guided me through the layout of the Crab Project and the plan of how it will all be performed. This is good becuase this is my first quarter of graduate school and I had only heard about the crab project around Thanksgiving of 2017.
Today I tested out Qubit for the first time as well as moving on to step 4 in the DIA protocol.
I tested out the RNA isolation protocol for the first time today, as well as have run into some difficulty in re-doing the DIA protocol.
Updates on RNA isolation; Podcast thoughts and subject matter thoughts; restarting the DIA analysis with the correct files.
Tommorrow I will be trying out RNA isolation on 2 extra Tanner crab hemolymph samples. Try protocol with 2 samples: 465 and 468 Both samples are yellow (from third sampling day) and are from crabs that are infected, immature, and held at ambient temperatures throughout the experiment.
Picking some preliminary samples for RNA isolation and getting questions answered about the RNA isolation protocol.
Notes from a meeting on DIA analysis and the 2015 oysterseed project with Emma, Steven, and Kaitlyn.
Ordering the mics for the Podcast and this week I will test out the RNA isolation protocol.
Testing out the Podcast and how the microphone, garage band, and publishing works.
I would have made this post on the actual first day of February, but I have been sick - the neverending cold. Anyway, for this short month I have some goals I’d like to achieve:
I prep for RNA isolation and understanding the protocol, and continue with the 2015 Oysterseed DIA.
Initial info on making a Podcast from Audacity workshop at UW and Crab Hemolymph samples next steps - testing out the RNA isolation protocol.
Today I chose some crab samples for RNA extraction. I made a chart with the tube numbers for the samples. All crabs are immature males. Due to a massive die-off in the warm temperature treatment groups, we unfortunately only have two infected and one uninfected sample per the final two sample days. As a result, the uninfected samples will not be “pooled”.
New qPCR machine arrived, training with Brian, and preliminary crab hemolymph sample picking for RNA isolation.
I posted the crab data from Pam to Owl, and I hear back from Emma in regards to my error rates.
Finished determining error rate for the DIA analysis and sent my results to Emma. Also, include some background info on the 2015 Oysterseed project.
Update on error rate peak picking - confusion as to how to categorize correct and incorrect picks by Skyline. Includes tips from Laura.
Update on DIA anaylsis of the 2015 Oysterseed project, working on determining the error rate of Skyline’s peak picking.
Dates and links to my old notebook posts on when I spun down the hemolymph samples from Pam.