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…
When I arrived today, there was a “fatal error” in Walnut for February RAW files number 7 and 8. So I tried it again, but it still didn’t work.
So then I went back to owl and re-downloaded the two RAW files. I then converted them to .mzML using MSConvert. Then I added them to Walnut, and it worked! Then I hit “Save BLIB”.
Then, I went through the DIA protocol, but I made a few changes per Emma’s suggestions.
- I used the new BLIB file I made using Walnut
- In Step 4e (12), I changed the minutes to 5
- In Step 4f I made sure I used the same .mzML files as the results that I used to create the BLIB in Walnut
So, with all this changes, the peaks still looked awful. I didn’t calculate an error rate because it felt like a waste of time. Instead I just looked at about 15 or 20 random peptides and noted that not one of them looked good. There was tons of noise and no clear peaks. I don’t know what to do, so I emailed Emma and sent her a .zip of my Skyline document. I JUST sent it a little bit ago (around 4pm), and I know she’s leaving for a bit, so she may not see it for a while, unfortunately.
I know Nick from Skyline suggested I use the Advanced Peak Picking option, but the protocol doesn’t have super clear instructions for how to do this for DIA Analysis. Emma told me that the pipeline for this isn’t super concrete yet, so that may be why I’m not sure how to do it.
I’ll leave this for now and go back to it on Monday with some fresh eyes, and maybe a response from Emma…
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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.
Walnut is an upgraded PECAN and is used to create the BLIB file which I will use in my DIA analysis of the 2015 Oysterseed RAW files in Skyline.
I converted the RAW files to mzML using MSConvert. Then, I uploaded those files to Walnut. Will take a long time so I’ll leave them alone and come back to them tomorrow morning.
NPRB Progress Report
I called Pam this morning to go over the progress report which is due by July 31st (Tuesday). Unfortunately we’ll have to change/add some things to it regarding the issue with the current pools as detailed in my previous notebook post: Crab Pools and Skyline Update. My post is from right before I left for a little vacation to visit family. So, Sam tried to take a closer look at what is going on.
Here’s his post: RNA Cleanup – Tanner Crab RNA Pools
Essentially he used the RNeasy Plus Mini Kit (Qiagen) on the three pools. Then he ran Qubit and Nanodrop1000 with the three pools. The results were similar to what he and I found the day before in that they were not good. The Nanodrop did not detect RNA in the pools. The Qubit didn’t detect RNA in Pool 1, and had very low numbers (~84ng of RNA in each pool) for pools 2 and the MasterPool.
Our target for sequencing is to have 1000ng of RNA in each pool, which total volumes of 50 ul. We are not there.
So, Sam is thinking of potentially having us try using RNeasy cleanup kit on some “test” samples to see if we can get better Qubit readings.
I am gonig to talk about this on Monday with Sam and Steven in person. I will either have Pam call in or update her later. And we’ll have to update the progress report, and I’ll have to do more labwork to try to figure out why these numbers are so low and how we can get these pools to where we need them to be. Stay tuned…
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