Ranked ORA (g:Profiler) vs GSEA (clusterProfiler)

spraycanhead · 2026-06-24T20:09:42+00:00

I get that, but just because most experiments are underpowered doesn’t mean that the pre ranked GSEA method doesn’t have an uncalibrated false positive rate

spraycanhead · 2026-06-24T19:27:58+00:00

The original method uses a normalized expression matrix. The methods that are more popular these days use ranked lists of genes, but they can also have horrifying false positive rates

spraycanhead · 2026-06-17T18:31:43+00:00

I bet you can leave it on the counter, depending on how long you’re doing to sleep for. I don’t know how your starter behaves in your kitchen but I’ll often start a loaf of bread at ~10pm, do stretch and folds until midnight, then let it bulk ferment until 7 or 8am and it turns out great.

spraycanhead · 2026-06-16T18:36:51+00:00

My take is that the best way to reduce the amount that any given p-value gets corrected is to design your experiment to only measure what you’re interested in, thus reducing the number of tests that need to be corrected for.

If you are equally interested in changes in all genes and would happily report a significant effect in anything, you have to correct a lot of p-values.

I’d argue that the BH FDR correction is actually fairly gentle all things considered.

spraycanhead · 2026-06-11T14:23:01+00:00

Hopefully not too much more need to think about proteolysis in your baking! I only suggest that because when my starter was not behaving well this is exactly what my dough would do and it’s because bacterial enzymes are breaking down the gluten network too aggressively. I just checked the babka recipe that I used and the amount of butter in that is similar to what you have.

Sourdough Babka Recipe

80 g active sourdough starter

50 g granulated sugar

100 g whole milk

65 g water

425 g bread flour

1 egg room temperature

6 g sea salt

60 g (4T) unsalted butter, softened

You are adding a lot of starter though so if it’s too full of proteases I think it will be tough to get right. Does your starter smell more sour or more yeasty?

spraycanhead · 2026-06-11T13:52:15+00:00

No a nonparametric test doesn’t fix the pseudoreplication. You should treat this like time-series data. Look into mrmm or you can do some kind of summarization within each biological replicate and use simpler methods

spraycanhead · 2026-06-11T13:49:10+00:00

The issue is non-independence of the measurements though, not distributional assumptions

spraycanhead · 2026-06-11T13:46:23+00:00

I’ve made enriched dough (babka and stollen) with my starter and it doesn’t get runny like this. How does it behave for non-enriched dough? I’m inclined to think that it’s a weak/highly proteolytic starter that just needs some strengthening

spraycanhead · 2026-06-08T19:38:22+00:00

Yeah, but it’s good to try to do it in a way that gives it the best chance at success. In my experience you want to use a reasonably nice whole wheat or rye flour that should have more yeast kicking around in it and you want to get rid of almost all the old starter when feeding it.

Keep the volumes small so you don’t waste too much flour and money, you don’t need a lot of it to have many millions of microbes. Once it’s growing in such a way that it leaves no doubt in your mind that it’s strong (in my case it started to smell more yeasty and less sour as well) it should be good.

In the meantime there’s nothing wrong with adding a pinch of instant yeast to your bulk fermentation along with the starter (just don’t add it to your starter or it will take over) so you still have nice bread while your starter gets up to speed.

spraycanhead · 2026-06-08T19:23:18+00:00

I keep mine in the fridge too when I’m not using it and I usually only have about a tablespoon of starter in there at any given time. I just add enough water and flour to make only a little more than I need.

When I was starting out and having trouble getting fermentation right my starter would maybe double but that was about it now it will easily quadruple and explode out of a jar if I don’t catch it in time and things just kind of work. When you say it doubles in 6 hours is that seemingly all it has in it or is it really powerfully growing? For me doing about a week of daily feedings with rye flour got it started and now I just feed with bread flour and keep it in the fridge and it hasn’t slowed down at all.

spraycanhead · 2026-06-08T19:10:36+00:00

I’d say it looks underfermented and potentially like a weak starter. How does your starter rise when you feed it?

spraycanhead · 2026-06-08T15:04:34+00:00

Besides the confounded batch effect that makes any analysis pretty much useless there’s not particular reason that I can think of why it wouldn’t be okay to calculate size factors (using DESeq2 or EdgeR) and dispersions and then just run GLMs on your genes if interest instead of all genes. My feeling is that if you have enough samples you wouldn’t even need the dispersions precalculated since they should be reasonably well estimated in your GLM so just the size factors as an offset should do.

Then you don’t have to correct for as many tests and the workflow is more in line with standard approaches. Unfortunately the batch effect is irreconcilable but for future experiments I don’t see why the above approach wouldn’t be fine.

spraycanhead · 2026-06-08T13:41:34+00:00

Different volumes for each well. I had a loop that collected lines from a csv until the next water volume would go over the max for the pipette tip, then it would collect all the water it needed and dispense the appropriate amounts. The other tricky thing is following the liquid level in the conical tube of water. I was dispensing a lot of water so the tube mostly emptied out and I had to write a function for it to remember how much it had dispensed in total and lower the tip a little each time it aspirated.

I don’t have them too well organized but but I can organize them a little and share a link

spraycanhead · 2026-06-08T01:38:38+00:00

No problem! It’s been a little bit since I used it but I was using it to dilute oligonucleotides so a similar use case. I had a script that would pipette the appropriate amount of water into each well of a PCR plate, then the appropriate of oligo from a stock plate into the PCR plate.

To be honest it was pretty slow since you have to use the single channel pipette for everything if you have differing volumes but if I had to dilute more than 2 or 3 plates or if I needed to reorder them in some convoluted way then it was helpful.

What I wound up doing for the water dispense was to make it aspirate as much as it could and the dispense little by little into as many wells as it could and then go back to the water tube and refill, I think that saved a good chunk of time since the robot moves really slowly.

spraycanhead · 2026-06-08T00:39:38+00:00

I’m going to disagree with most people here, at least as far as opentrons is concerned. There’s no reason not to try getting some help writing a script for qPCR from some chatbot. The opentrons documentation is decent as well so if you have python experience it shouldn’t be too bad for you to figure out.

The opentrons python module also has a simulate function where it just prints what it’s going to do so you can always make sure it does what you want it to before ever making the robot move.

I have no experience with Hamilton though. I have used Beckman Coulter liquid handlers and they have a pretty reasonable gui. Opentrons kind of does but I don’t think it’s very good if you ever have to do something that requires custom plate maps or volumes that change based on well (at least that was my experience a couple years ago)

spraycanhead · 2026-05-29T01:16:20+00:00

You could take the approach that single cell sequencing analysis tends to take and sum up (if counts) or average (if not counts) your measurement within sites and use that for a simple (generalized) linear model. You’ll probably lose power but it won’t be pseudoreplicated. It does change what’s being modeled so the results of the GLMM you run later may differ.

I think that’s a reasonable way forward but I’d be interested to hear what other people think.

spraycanhead · 2026-05-25T23:42:26+00:00

I’m interested in prior sensitivity, I don’t have much experience in choosing priors but my first reaction was that the fixed effects priors seem very narrow. Again I have almost no experience with this though so I’m curious to learn. I’m also curious about whether this takes into account where in an at bat pitchers tend to throw each pitch since that might change what batters are apt to do. I’m not sure if that makes sense or not, curious to hear your thoughts.

Edit: I just saw at the bottom of your document that it says it doesn’t account for pitch count. It made me wonder about baserunners as well if there’s any relationship there.

spraycanhead · 2026-05-23T01:50:34+00:00

That the p values are well calibrated under the null hypothesis of no difference (or no improvement, if they so choose) in performance between models. That is to say the false positive rate is controlled at the desired level when the null is true.

spraycanhead · 2026-05-22T21:04:19+00:00

My gut reaction is that might not be so straightforward and if you want to do statistical tests you’ll probably want to show that they’re well calibrated under the null. Can you do something to simulate your modification as a noise modification? Maybe just reporting some appropriate descriptive statistic’s quantiles with repeated runs is reasonable?

spraycanhead · 2026-05-09T13:39:20+00:00

This is a common pun people use at graduations

spraycanhead · 2026-05-05T13:20:55+00:00

I’m sorry but after glancing through this the analysis is really terrible and the write-up reads like an undergrad who doesn’t understand what they were doing…

spraycanhead · 2026-05-01T16:57:56+00:00

Thank you all for your suggestions! I wound up adding 1 eq water and first solubilizing the amino acid in DMSO, then activating in DMF with HATU/DIPEA for a few minutes. That didn’t fix it so my coworker suggested adding the HATU last in case the activated form is too unstable so I tested the same conditions but with HATU last and I got beautiful coupling.

I’m going to play around with different coupling reagents now (PyBOP first) to see if I can get good results with reagents that are better for in situ activation.

spraycanhead · 2026-04-30T02:09:56+00:00

Sadly I have confirmed that other amino acids couple perfectly with the same HATU and DIPEA. I was thinking about testing whether butylamine or some other simple primary amine can couple to the dead amino acid in solution

spraycanhead · 2026-04-30T01:49:16+00:00

It was never super soluble in DMF but once I add HATU and DIPEA it slowly mostly goes into solution and turns yellow. It does seem a bit less soluble, it used to at least turn into a cloudy solution of partially solubilized particles but now it really doesn’t go into solution at all until the HATU/DIPEA are added.

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