Laura’s Notebook: Cleaning Srm Data In Skyline

First steps, done on 8/8

Clean up data in Skyline:

Used final version of sequence file (which I uploaded from the Vantage computer to Owl @ UWPR on 7/28) to associate .raw files with samples – determine which samples are which for the duplicate named files. The final sequence file, which will also contain the time loaded into tray & injection time (in case this is a factor), is located in the Data folder of this repo, here.
image

Make sure I have all data in Skyline

Confirmed in Skyline by going to Edit -> Manage Results & cross-checking data file names in Skyline with the sequence file, and the .raw files on Owl. image

Confirm correct peaks picked & determine if any transitions/peptides are un-usable

Referenced the Predicted SRM Retention Time calculations I did at the beginning of my mass spec run to confirm correct peaks picked. ID’d peptides with lots of noice, and no clear peak at the predicted RT. Here is a screen shot of the predicted RT, with poor quality peptides in red. NOTE: Need at least 2 transitions for each peptide to move forward with analysis. image

Next steps:

  • Adjust peak boundaries
  • Determine if any .raw files should be discarded, based on poor-quality data and those that I re-ran & re-made
  • Look @ all blank runs to see if there are any weird signals
  • Check out blank samples
  • Download data! Protein, peptide, transition, area, retention time
  • Work through Emma’s suggestions: normalization, dilution durve check, NMDS, ROC curves, data upload

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Grace’s Notebook: August 8, 2017

Manchester Monday:

Yesterday I assisted Yaamini at Manchester with her gigas larvae. It was a similar process to what was done with Laura’s, however Yaamini’s are not in a flow-through water system.

I screened larvae at 60 µm. 24 buckets.

Yaamini sampled, killed with googols, and counted larvae.

Olivia shuffled buckets, cleaned, and kept things organized.

I will be doing Yaamini’s counting job on Friday and Sunday, while she is out of town. I will have help from two of Joth’s helpers Friday, and Kaitlyn will come out with me Sunday.

Today:

Today is Day 2 of Lab HackWeek. Steven, Kaitlyn, and I cleaned up FTR 213.

My other tasks included:

  • Creating a Lab Label that can be put on our equipment that we move to places like Manchester (made 1×1 and 2×2 inch: here)
  • Screen Shot 2017-08-08 at 4.00.46 PM

  • Researching options for searchable and public image sharing for lab images – Steven would prefer using something Google-related

Yaamini’s Notebook: Gigas Larvae Day 8

Immersion heaters are pesky

Overall, today was pretty smooth. Olivia and Grace helped me screen my larvae. At first, I thought I would be able to screen on both an 80 micron and 60 micron screen. However, after screening the first two buckets, Bucket 11 and 6, I didn’t count any larvae from the 80 micron screen. I decided to continue just screening on a 60 micron screen, and try again with two screens on Wednesday. I did something different today where I added food to the bucket before restocking, instead of adding food to all buckets at the end. I think this method is better because the larvae get fresh algae after being stressed by the screening process, so I’ll continue to do this. While screening Bucket 12, Grace accidentally screened through a 48 micron screen first. She caught her mistake very quickly, but it’s possible some larvae were lost with this additional screen. All of my larvae are at about 2 larvae/mL, which is the stocking density I want at this point. You can see my counts and feeding log here.

Now here’s the annoying part. Totes 3, 4, 5, 7 and 9 all had their high alarms (27ºC) go off after the buckets were restocked. Tote 4 and 7 had their alarms go off multiple times! To reduce temperature, we used tripours to drain out some of the water in the tote, and then we refilled with ambient freshwater instead of using the water from the heating reservoir line. This quickly brought our temperatures down. I think refilling totes with ambient freshwater should be our standard now. Adding heated water to a tote that’s already at temperature may set it over the edge.

For tomorrow:

  • Record tote positions
  • Add new HOBO loggers buckets without them
  • Feed oysters
  • Finalize help schedule for this weekend
  • Prepare task lists for those helping

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