Kind of. Using the digested proteome solved my problems of not having valid MS2 data. However, Sean reported that we’ve hit a new snag. It seems like the memory request argument isn’t a global memory request, but specifies memory for each instance of
pecanpie. Basically, the first instance takes up all of the memory and the following instances time out. Sean’s suggestion is to rerun
pecanpie and tell it to only look at three isolation windows concurrently as it makes its way down the isolation scheme.
In the meantime, I’m going to draft poster layouts and modify the DIA wiki and our DNR Paper. Seriously hoping things work before I go out of town Wednesday!
Figure 1. #MOOD
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We’re officially halfway through our pH treatment window!
Laura and I did the following today:
- Water chemistry sampling: 1L grab samples and bottle samples
- I siphoned fecal matter out of tanks while Laura rinsed the Olys with freshwater
- Cleaned filters
- Bleached algal lines
- Modification to procedure: 20 mL of bleach into 6 L of water, then run a full bucket of freshwater through to ensure bleach is completely washed through lines
- Laura installed a water alarm in the large header tank, but the water is so turblent in there that it doesn’t work the way we want it to
- Moved probes between tanks
When cleaning oysters, I found a completely dead (not mostly dead C. gigas in Tank 3. I could tell it was dead because it was gaping even when out of the water.
Figure 1. Dead oyster from tank 3 (left) next to another alive oyster (right). Oysters that are dead will continue to gape out of the water when compared with their living counterparts.
We also found a couple of fat and happy polychaete worms on a few oysters. My hands were really wet at the time so I couldn’t snap a picture, but I did manage to get a photo of another baby anemone I found on an oyster. I’m not sure where these polychaetes came from, since we thoroughly cleaned the oysters last week.
Figure 2. Baby anemone found on an oyster. This is the second anemone I have found.
PSRF upped the feeding to 500 mL of Reed’s Shellfish Diet per day (roughly 2 billion cells per milliliter spread out between six culture tanks and the two larger C. gigas tanks on the bottom). The tanks were a little too dark and we think the extra algae might be modifying water chemistry, so we asked them to drop it down to 450 mL daily.
Things I need to do by Monday:
- Update chemistry data spreadsheet
- Get PSRF to poison samples while Laura and I are at NSF
- Write up a protocol for Grace and Olivia to follow on 3/29
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So it looks like we got the pecan workflow to work on a subset of isolation windows and samples. I also learned a few more things about `pecanpie` during the process.
pecanpie adds a
-Q argument to the
percolator command run on each sample. According to the
percolator documentation, this argument does not exist and results in an error and a failed run. After removing it from the
percolator` job file it works fine.
2. This is likely only an issue with toy/shortened data sets, but percolator is sensitive to the number of true positives available to populate a training data set, if it doesn't have enough to detect directionality in the trend it throws an error asking for changing the False Detection Rate to something greater than the stock 0.01 via a
-F argument. This doesn't quite work as there are two methods to change FDR rate, the
-F flag as mentioned in the error message as well as a
-t found via a GitHub repo issue which is not mentioned in the error message.
3. This is a big one for quality of life. the
--pecanMemoryRequest argument from
pecanpie is apparently for a single instance of pecan, so if you allow 12 instances to run, the actual memory requested is
number of instances *
--pecanMemoryRequest. I found this out the hard way when I thought I allowed RoadRunner to use 30gigs of the 48 gigs of memory available for the pecan runs. I instead let it use 30 gigs * 12 instances of memory. Oops.
I also met with Hollie today and got some plans worked out for near term Geoduck methylation analysis. I made a 10x coverage whole genome methylation file for all samples, as well as started working on a Circos plot for the genome. The Circos plot is going to take some tinkering, as they're generally built around loci found in some small number of chromosomes as opposed to loci found in 40,000 scaffolds. We also decided to look at the beta-binomial model implemented in RadMeth to compare to the results from MACAU.
All in all, plenty of things to work on over the next week or so!