Capturing expression in Canto (with implications for website allele /genotype)

[ValWood]

1. The ability to specify the plasmid promoter (mostly of these are stored in names, background and comments)

2. If overexpression is selected, the ability to specify the "fold" overexpression (I.e 2X, 10X, 100X)
We probably need to discuss the details.
We need this to
a) be able to distinguish different expression levels (and hence phenotypes) for overexpression alleles (and knockdowns)
b) to clean up backgrounds and comments
c) To fix some allele names which do not follow standard naming conventions
d) also ectopic expression

example
Check cut12-s11
https://www.pombase.org/genotype/cut12.s11-G71V-amino_acid_mutation-expression-not_assayed
https://www.pombase.org/genotype/cut12.s11-G71V-amino_acid_mutation-expression-wild_type_product_level

[manulera]

Following up on the discussion with @ValWood today. Perhaps for the phenotype annotations it is not so important, but when querying for alleles in Intermine I think it would be good to add a category "promoter" to alleles (NULL if not changed) in addition to "Allele expression".

Overexpression or knockdown are often induced in certain conditions, such as removal/addition of thiamine for nmt1 promoters, but I think the allele description should represent the changes in the DNA / protein. Same is true for ectopic expression (e.g. for inducing expression of meiotic proteins during another phase, or vice versa).

(Maybe this belongs in another issue, but somewhat related to this)

[manulera]

Hello,

Yes, mostly that, but I think the (4x) or (20x) should go in the allele expression column, not in the promoter, since the expression levels will depend on experimental conditions for a given promoter.

For example, It is not very common, but sometimes you see that people use nmt41-GFP-gene constructs just to tag proteins in their N-term. They then do the experiments in presence of thiamine (overexpression not induced). These nmt1 promoters are quite leaky, so even in the presence of thiamine they can produce equal or higher levels of protein than the wild type. nmt1 always that I have used it produces an overexpression even when repressed, nmt41 not so sure.

If used for a localisation experiment, people probably will not test whether the expression levels are higher than if GFP-gene was expressed from the normal promoter. I think this is mostly done to quickly tag a protein in the N-term when a C-term tag would be lethal or perturb the behaviour of the protein.

[ValWood]

@manulera says:

It was in my todo list to make an issue about this exactly. I think the problem is the ambiguous use of expression levels and alleles. We discussed this briefly before. I think allele description should be only concerned with describing the DNA sequence, and expression level should be a different field since it is conditional.

We could make promoter_changed a type of allele, and we could indicate which promoter has been used when it is known. Also, a change in promoter alone may not lead to a change in expression (it may need to be induced or repressed), but typically if a phenotype has been described it is safe to assume that the experiment has been conducted at conditions where the expression levels change.

You can see what I mean with the following query, you see that all alleles that come up in wild_type are from those that come from changes in expression levels (see that the only values are knockdown and overexpression):

[ValWood]

we don't need
#2629
becasue of this proposal.

[ValWood]

Here are some examples of expression differences that need differentiating:
https://curation.pombase.org/dumps/latest_build/logs/log.2023-04-04-21-42-39.chado_checks.duplicate_allele_descriptions

[for now we won't worry about the copy number type overexpression, we can get these into Chado somehow, they are edge cases)

[ValWood]

DECISION:

i) An extra option for ectopic expression - moved to #2723

ii) Add additonal dialogue If you select Overexpression, Knockdown or Ectopic
Pombe gene promoter [text box] -> pick a gene
Exogenous promoter [ free text and autocomplete from existing]

iii) Store promoter info in Chado - moved to pombase/pombase-chado#1087

mock up; )

[ValWood]

I put this at high priority, since it will be useful to finish up all of the allele QC work.

[ValWood]

Hi Kim,
Sorry for the dealy on this one. We can change the text to "promoter swap (fission yeast)"
and make this live

[kimrutherford]

Sorry for the dealy on this one. We can change the text to "promoter swap (fission yeast)" and make this live

I've merged the code into the branch. It took a while because there were a lot of conflicts to sort out. I'll deploy the change when oliver1 is back.

[kimrutherford]

"promoter swap (fission yeast)"

Would "promoter swap (Schizosaccharomyces pombe)" or "promoter swap (S. pombe)" be OK? We don't have "fission yeast" in the config or in the Canto database (this has to work for non-pombe Canto instances too).

[kimrutherford]

I've merged the code into the branch

I meant, I've merged the promoter branch into the main branch.

[kimrutherford]

Would "promoter swap (Schizosaccharomyces pombe)" or "promoter swap (S. pombe)" be OK?

[ValWood]

Would "promoter swap (Schizosaccharomyces pombe)" or "promoter swap (S. pombe)" be OK?

yep

[kimrutherford]

OK, great. I've implemented that ready for when oliver1 is back.

[ValWood]

@PCarme and I tried to use the new promoter feature but we couldn't see it?

[kimrutherford]

Ah, sorry. I never got around to updating the main Canto with the change. We will need 10-20 minutes of down time to do the upgrade.

[ValWood]

OK, lets wait until the weekend (maybe your Monday morning), because I sent tonnes of sessions out today. We are already getting a few back.... don't want to disrupt their flow!

[kimrutherford]

That makes sense. I'll update when it's quiet. First thing Monday morning NZ is a good time.

[kimrutherford]

I never got around to updating the main Canto with the change. We will need 10-20 minutes of down time to do the upgrade.

That's done now.

We should discuss how the promoters should look in the genotype list.

[ValWood]

I'm going to do this session now and we can use as an example

mad1/~10%,SPBC3D6.04c:allele-28,nmt81 promoter,{ed9652a888c24c94}
mad1/~30%,SPBC3D6.04c:allele-29,bub3 promoter,{ed9652a888c24c94}
mad1/~300%,SPBC3D6.04c:allele-31,ark1 promoter,{ed9652a888c24c94}
mad1/~500%,SPBC3D6.04c:allele-30,nmt1 promoter,{ed9652a888c24c94}
mad2/~10%,SPBC20F10.06:allele-14,mad3 promoter,{ed9652a888c24c94}
mad2/~65%,SPBC20F10.06:allele-11,hphNT1 cassette in promoter,{ed9652a888c24c94}
mad3/~30%,SPCC1795.01c:allele-10,hphNT1 cassette in promoter,{ed9652a888c24c94}

[ValWood]

I've done this one, it looks like this in Canto genotype list:

which is fine?

[kimrutherford]

Yep, that's how it looks for now. I'll check Chado in the morning.