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[Trekkin]

Well, are they still asking about Sanger sequencing, or is it all pyrosequencing/nanopore/Illumina now?

Oh no, there's so much more than that in this part of our module that come under the sort of genetic analysis/sequencing umbrella.

PCR (obviously), next-gen sequencing, sanger, RFLP, RAD-Seq, GBS, SSR, KASP, QTL linkage mapping, and GWAS are the ones in our lectures. We have to memorise all of them in case they turn up in an exam. That's nine things I can't remember. I only remember PCR, and I don't remember it that well. Especially not the temperatures.

The thing I really hate about this is that they're acting as if we're going to need to do some of this stuff without access to any sort of resource that lets us understand what's going on. I mean, yeah, I'm gonna wind up doing fucking RAD-Seq after the nukes have fallen and I've lost the ability to read and hear due to some very specific brain damage.

Oh, this one of my pet peeves. There's a perfectly good conceptual structure here that everyone discards in favor of a flat list of techniques.

See, there's only one way to amplify DNA, and it's PCR; NGS, Sanger, RAD-SEQ, GBS, and KASP are all PCR-based, just with different reaction conditions. Likewise, every way to read DNA is some variation on "different bases are going to glow different colors"; there's some variance in how we extract length information, but if you keep that requirement in mind, a lot of the faffing about with bridge PCR and barcodes and beads and emulsions starts to make sense. Then you can describe the techniques in terms of each other, and the associations should help it stick in your head.

Sanger, for example, is PCR spiked with labeled dideoxyNTPs, so it's going to terminate the PCR reaction and leave the last base labeled. Then we just run a gel and watch the sequence of last bases. RAD-Seq is parallelized Sanger, but we prep the sample by restriciton digest first and our primers are all RAD tags unless we ligate on universal adaptor barcodes. NGS is RAD-Seq, but we sort the fragments from the RAD-Seq prep and run Sanger-esque seq-by-glowy-PCR on all the fragments simultaneously on a flow chip or beads or in an emulsion or something. The physical separation almost doesn't matter; it's just literally massively parallel barcoded Sanger with some mechanism to localize amplification so you get one signal from each spot. GBS is ratchet NGS-lite with a specific purpose, which skews the digest panel from the RAD-Seq part so we get rid of the repeats and focus on the SNPs we care about. KASP goes one step further and modifies the primer selection too. QTL mapping and GWAS are just what you do with KASP/GBS/NGS data.

Once you think in terms of selecting different techniques through a modular workflow, I find it helps a lot of the methodological differences make more sense as exigencies of the techniques used in previous steps.

[dragdeler]

-snip-

[Parsely]

This thread makes me sad, why do I click on this thread?

[JoshuaFH]

This thread makes me sad, why do I click on this thread?

Welcome to the Sad Thread. We have Ritz crackers and a fountain dispenser with unlimited tears. All free!

[Arx]

I spent most of this afternoon and yesterday afternoon sleeping. I don't think it can be helped, between the stifling summer heat and my messed-up chemistry, but I hate it. I just don't have motivation to do other things, and I just end up wasting so much time.

[Yoink]

I can't tell where my current physical illness ends and my anguish at having guests staying begins.   
Am I not eating because sick, or am I not eating because those people are sitting in the kitchen, and to do anything in there would mean being forced to interact with them in at least some way?

Also had an exceedingly unpleasant phone conversation with my mother, who was seemingly doing her level best to make me feel even worse than I do already. I have social obligations and a doctor's appointment tomorrow and I am highly stressed about both of them.


Having a diarrhoea (wait, autocorrect says it's spelled that way? I coulda sworn I was told to spell it without the 'O') and headcold combo is unpleasant indeed. Just what was needed to stamp out any possible good I may have been starting to feel, I suppose.   

Happiness sure seems an impossible goal. I can't even fill any of its requisites.     

[Kagus]

Having a diarrhoea (wait, autocorrect says it's spelled that way? I coulda sworn I was told to spell it without the 'O')

It is and it isn't, much like how fetus is spelled foetus, or Jeff is spelled Geoff.


A quick spot o' Goog tells me that this is apparently a difference between British English and American English. And for some reason Australians seem to use American spellings for things, ya fuckin' druggos.

[scriver]

I have to insist Geoffrey is spelt like that for entirely different reasons also isn't Geoffrey pronounced Joffrey, not Jeffrey?

[Yoink]

And for some reason Australians seem to use American spellings for things, ya fuckin' druggos.

Wadiyatalkinabeet   

[JoshuaFH]

I'd stated that I hated myself, and I thought more deeply on it, and just pure, unchecked self-hatred is perhaps the neatest explanation for all my woes in life.

-How I frequently delete my posts, even objectively harmless ones, is because I hate myself and my own thoughts.
-How I agonize over every small mistake, is because I hate myself and want myself to adhere to a childish fantasy of perfection.
-How I have nearly no friends, and absolutely no lovers, is because I hate myself and consequently don't want to share myself with anyone in the world.
-How I hate even things I like, because they bring me pleasure and therefore must be construed in a negative light as being bad for me somehow.
-How it seems so impossible to remain disciplined in any self-improvement plan, as improving myself and hating myself are mutually exclusive, and my self-hatred won't go down without a fight.

and so on and so forth. It's not exactly a big revelation, but it's a recontextualization.

I saw my therapist today, it was a good session I think. I didn't have this theory on myself in time to share it with her, but I guess I have another session in 2-ish weeks.

[ChairmanPoo]

How I frequently delete my posts, even objectively harmless ones

Nothing is harmless anymore. This is a good idea. I wish we had a "purgue history" function.

Anyways I'm sad cause I'm leaving my home and flying back to my workplace today

[Yoink]

Somebody posted this on Facebook and, while I like to think I'd come to most of these realisations on my own, it's still a good read.

I feel like some of y'all in this thread might get something from it as well, if you like. Maybe. It's Wil Wheaton talking about his struggles with mental illness.
I'm not entirely sure why I'm posting this here, I mean, it's highly doubtful that this is the best, most helpful piece ever written on the subject or anything, but... well, it's Wil Wheaton, everybody knows him, plus I was halfway through typing this drivel before I realised the entire post was probably a dumb idea.
So, waste not want not and all that. Can't be just throwing away precious used keystrokes.   

[Dunamisdeos]

That's a really good read, thanks for posting it.

[Rockeater]

I finished the first semester of my master's degree, I would've wrote it in the happy thread if not for the fact that the last lecture and the last few lectures in one of the two courses I took this semester was undispherble to me, there were entire boards of symbols where ignoring the ones who were in requirements courses I only understood the meaning of 5 or less, probably less, the worst part is that I might been able to understand them if I read the material in home, which I didn't because of a combination of my laziness and the fact that every day in the army left me mentally drained from boring job and stress about things people insisting I know how to do even though no one explained to me, at least it's a test done from home so I have 3 months to finish with it.

Well that was me rambling waiting for a train.

Edit: And now being pretty sure I have depression or anxiety and reading the above article and recognising some of his discriptions of depression and anxiety in myself I still feel like I just try to find excuses for myself for being lazy and not social, woohoo.

Sorry on the venting

[Kagus]

was undispherble to me

That word is undispherble...