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Isolated Yeast (Tree House): How to Identify and Characterize?

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isomerization

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Long time lurker, finally decided to be productive and join the community.

So I am a biochemist/structural biologist (bacterial pathogenesis) and I love making/drinking IPAs (homebrewing for 2+ years) of all kinds.

I was drinking a Tree House Julius with a buddy of mine and we decided that we'd figure out what kind of yeast they use. So I harvested the dregs, got them growing and then plated out serial dilutions for isolation (single plate shown):



Using a PCR method (https://suigenerisbrewing.blogspot....howComment=1490284392639#c8540474511060838192), I was able to amplify the ITS region (http://sites.biology.duke.edu/fungi/mycolab/primers.htm) of this yeast (my band is ~800 bp):



I then sequenced the amplicon, and used BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to search for similar deposited sequences. I think this is where I've run into a wall, as the best hits I get back are only ~89% identical (they are S. cerevisiae). Last year White Labs (and others) published a really cool paper (http://www.cell.com/action/showMethods?pii=S0092-8674(16)31071-6) where they whole genome sequenced 157 S. cerevisiae yeasts. Those sequences are deposited, but I think my BLAST search is missing them for some reason.

Anyways, I am moving on the phenotype side of characterizing this yeast. Can anyone suggest experiments (lab side or fermentation) to figure more out about this isolate? I have a session NE IPA planned in the next couple weeks and can update more once that's done.
 

xerotime

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I've been considered that the Treehouse yeast may be a blend of 2 different strains. Could that be why you are running into trouble identifying? It seems odd with all of these newer small yeast labs (Giga, Yeast Bay, etc.) that no one has grown it up for sale like what has been done with Conan.
 
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isomerization

isomerization

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My only hold up with considering that they use a blend of yeast for fermentation is that would (seemingly) be really difficult to maintain over time. I'd expect growth rates to throw off the ratios over time and result in different fermentation/flavor/etc profiles. Something that is more likely (in my mind) is that they are using a genetic cross of two strains, something like the Conan x WLP644 strains (discussed here https://www.homebrewtalk.com/showthread.php?t=577066).

That doesn't explain why, as you mentioned, there haven't been any attempts to sell this strain by a commercial yeast lab. I've seen several mentions on HBT and other blog sites of people cultivating yeast from TH cans. I didn't test individual yeast colonies for genetic diversity though.

My issue with identification is that I don't have access to the right database (I think). I've contacted some companies that do contract work for professional brewers, but the fees aren't worth the information ($200). While I hate waiting for information, the best course in this case is to do some split batches and compare the yeast I harvested against WY1318, Conan and the like.
 

TheHairyHop

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My only hold up with considering that they use a blend of yeast for fermentation is that would (seemingly) be really difficult to maintain over time. I'd expect growth rates to throw off the ratios over time and result in different fermentation/flavor/etc profiles. Something that is more likely (in my mind) is that they are using a genetic cross of two strains, something like the Conan x WLP644 strains (discussed here https://www.homebrewtalk.com/showthread.php?t=577066).

That doesn't explain why, as you mentioned, there haven't been any attempts to sell this strain by a commercial yeast lab. I've seen several mentions on HBT and other blog sites of people cultivating yeast from TH cans. I didn't test individual yeast colonies for genetic diversity though.

My issue with identification is that I don't have access to the right database (I think). I've contacted some companies that do contract work for professional brewers, but the fees aren't worth the information ($200). While I hate waiting for information, the best course in this case is to do some split batches and compare the yeast I harvested against WY1318, Conan and the like.
I don't think it's a hybrid. Treehouse started from really humble beginnings and they've been turning out the same beer, really. I think, if it would be a guarantee, you could find enough people to chip in for that $200. I would certainly be one of them
 

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Unfortunately you won't be able to do any strain-level identification with the ITS sequence; it is only good for species-level identification. If you are willing to share the sequence, I have a local BLAST database with the 157 whole genome sequences from the Cell paper you linked to, and could search it with your sequence. But as I mentioned, I would not trust strain-level identification based on the ITS sequence (little variation between strains and more importantly it is only a small single region in the genome. E.g. of the strains in the Cell paper, many have identical ITS sequences, but belong to different clades).

You could instead try interdelta primers or RAPD primers:
http://onlinelibrary.wiley.com/doi/10.1016/S0378-1097(03)00205-2/full

You could then compare the electrophoresis band profile of your isolate with those you obtain from some different control strains (e.g. WLP001, WY1318, Conan, etc.). That should give you better idea of what its closest relatives are. If possible, include as many control strains as you can.
 
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Unfortunately you won't be able to do any strain-level identification with the ITS sequence; it is only good for species-level identification. If you are willing to share the sequence, I have a local BLAST database with the 157 whole genome sequences from the Cell paper you linked to, and could search it with your sequence. But as I mentioned, I would not trust strain-level identification based on the ITS sequence (little variation between strains and more importantly it is only a small single region in the genome. E.g. of the strains in the Cell paper, many have identical ITS sequences, but belong to different clades).

You could instead try interdelta primers or RAPD primers:
http://onlinelibrary.wiley.com/doi/10.1016/S0378-1097(03)00205-2/full

You could then compare the electrophoresis band profile of your isolate with those you obtain from some different control strains (e.g. WLP001, WY1318, Conan, etc.). That should give you better idea of what its closest relatives are. If possible, include as many control strains as you can.
I was actually just going to email/PM you about this after reading through your blog post linked to the WLP644 x Conan thread.

I have the delta12 and delta 21 primers ordered, should arrive tomorrow. I have the following strains to test using this PCR method:

WY1056
WY1272
WY1318
WY1332
WY1968
WY3944
WLP802
Yeast Bay Vermont Ale
WLP670 (isolated each strain, one Saison and one Brett)
Julius isolate

If I remember correctly from the paper you cited, nothing should be amplified with the Brett strain, correct?

Once I have the gel image, would you be interested in helping interpret the results Suregork?
 
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Cainmos

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As an avid fan of Tree House and IPAs in general I approve of this work! Interested to see the results.
 
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isomerization

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I can't help with the BLAST results without the sequence or parameters, but I would recommend trying BLAT, by UCSC. I think they have a microbial version.

There is also
http://www.yeastgenome.org/blast-sgd

Looks like they use wublast.
As I am now aware of, the stretch of DNA I originally amplified and sequenced is only reliable for identification across, and not within, species of Sacc. I am working toward a new technique (for me) of DNA fingerprinting (see posts by suregork). Hopefully should have something in the next week or so to analyze.

With that said, I went back and looked at the DNA chromatogram a bit closer, and there is a suspicious amount of overlapping peaks. Normally this would imply heterogeneity in the sample that was sequenced, but I'm honestly not sure at this point. I had a clear single band by PCR (image in first post). I'm a bacteria guy, not a yeast biologist, so again not sure why that happened.

I will post the DNA sequence when I'm back at work tomorrow, and can send the DNA peak file to anyone that wants to look at it.
 
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isomerization

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UPDATE:

10 strains of Sacc have been struck out:



I then selected a single colony from each plate, grew the yeast in YPD for 24 hr at 30C and harvested the genomic DNA. Spectroscopic analysis indicated it was relatively pure and free from protein contamination:



Next week I will start on the DNA fingerprinting method and report back.
 
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isomerization

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I'd be happy to help! I recommend using a higher agarose concentration (e.g. 2.5%) to help separate the smaller bands.
I must admit, I almost gave up getting the PCR to work, but today was a good day (it is Friday after all)!

I stumbled across this technique in a paper (https://link.springer.com/article/10.1007/s10295-010-0837-z) as I was troubleshooting, instead of trying to isolate the DNA from a yeast culture, all you need to do is:
1.) pick a single colony (I suspect you could get away with a tiny amount of yeast from a liquid culture as well)
2.) drop it into sterile water in an eppendorf tube, close cap
3.) microwave on high for 5 min
4.) use 1 ul of this mixture in a PCR



Strain key is as follows:
1 - WY1056
2 - WY1272
3 - WY1332
4 - WY1318 (this culture did not like YPD media very well for some reason)
5 - WY1968
6 - WLP670 (saison isolate)
7 - WLP802
8 - Julius isolate
9 - The YeastBay Vermont Ale
10 - WY3944

Suregork, I am working on improving the quality of this image. Using less DNA dye (white bands across top and bottom of gel), more sample (15 ul here, but can load up to 50 ul on a larger gel), getting better bands for lanes 2 and 3, etc.

The only thing that really jumped out at me is WY1968 and Vermont Ale (aka Conan) have almost identical banding patterns! Any thoughts with the current image?
 
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I forgot to add that the Julius isolate is certainly unique via this DNA fingerprinting method!

So, what this really means is that I need more yeast samples to analyze, I'm looking into figuring out an easy way to mail samples through the postal service (only costs a stamp!), if anyone has suggestions and is interested in sending yeast that is not on my current image, please let me know!!
 

yourlastchance89

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Subbed! Always loved the biological aspect of brewing moreso than the chemistry or engineering that seems to encompass almost all homebrewing. Can't wait to see what you find out
 

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I must admit, I almost gave up getting the PCR to work, but today was a good day (it is Friday after all)!

I stumbled across this technique in a paper (https://link.springer.com/article/10.1007/s10295-010-0837-z) as I was troubleshooting, instead of trying to isolate the DNA from a yeast culture, all you need to do is:
1.) pick a single colony (I suspect you could get away with a tiny amount of yeast from a liquid culture as well)
2.) drop it into sterile water in an eppendorf tube, close cap
3.) microwave on high for 5 min
4.) use 1 ul of this mixture in a PCR



Strain key is as follows:
1 - WY1056
2 - WY1272
3 - WY1332
4 - WY1318 (this culture did not like YPD media very well for some reason)
5 - WY1968
6 - WLP670 (saison isolate)
7 - WLP802
8 - Julius isolate
9 - The YeastBay Vermont Ale
10 - WY3944

Suregork, I am working on improving the quality of this image. Using less DNA dye (white bands across top and bottom of gel), more sample (15 ul here, but can load up to 50 ul on a larger gel), getting better bands for lanes 2 and 3, etc.

The only thing that really jumped out at me is WY1968 and Vermont Ale (aka Conan) have almost identical banding patterns! Any thoughts with the current image?
 
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isomerization

isomerization

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Not that I have tried it, but I've read the following on the Sui Generis Blog about mailing yeast samples.

Mailing Yeast

New Mailer System
I should have thought of that since I enjoy his blog so much!

If anyone is interested (and capable), I am more than willing to mail you autoclaved blotting paper for you to spot down yeast and mail back to me. Send me a PM if you have multiple strains not on the list above and are interested in swapping. I'm happy to send what I have access to as well.
 

suregork

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I must admit, I almost gave up getting the PCR to work, but today was a good day (it is Friday after all)!

I stumbled across this technique in a paper (https://link.springer.com/article/10.1007/s10295-010-0837-z) as I was troubleshooting, instead of trying to isolate the DNA from a yeast culture, all you need to do is:
1.) pick a single colony (I suspect you could get away with a tiny amount of yeast from a liquid culture as well)
2.) drop it into sterile water in an eppendorf tube, close cap
3.) microwave on high for 5 min
4.) use 1 ul of this mixture in a PCR



Strain key is as follows:
1 - WY1056
2 - WY1272
3 - WY1332
4 - WY1318 (this culture did not like YPD media very well for some reason)
5 - WY1968
6 - WLP670 (saison isolate)
7 - WLP802
8 - Julius isolate
9 - The YeastBay Vermont Ale
10 - WY3944

Suregork, I am working on improving the quality of this image. Using less DNA dye (white bands across top and bottom of gel), more sample (15 ul here, but can load up to 50 ul on a larger gel), getting better bands for lanes 2 and 3, etc.

The only thing that really jumped out at me is WY1968 and Vermont Ale (aka Conan) have almost identical banding patterns! Any thoughts with the current image?
Will have a closer look at this tomorrow, but based on a quick glance it seems like your isolate is distinct from the other strains you've included and WY1056 is the closest relative. Interesting yes that the Vermont Ale and WY1968 seem closely related :)
 
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isomerization

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First test using the isolated Julius yeast is done. Same recipes were used (albeit one week apart, so caveat there for sensory analysis below) and one batch was fermented with WY1318 while the other with the Julius isolate described here.



Left is WY1318 and Right is Julius (poured from growlers that were filled earlier in the day, otherwise they have similar head retention). I stupidly didn't fill the glasses up evenly, but the color and haze appear pretty much identical to my eye. Both beers had the same OG (1.058) and almost the same FG (1.015 and 1.014 for WY1318 and Julius isolate, respectively. Beers were mashed at 156 F. If I mash lower (151 F) for WY1318, it usually drys out too much (1.010-1.012 range) for my tastes with this recipe (h/t Braufessor).

I'm terrible with flavor descriptors, but I'd say the biggest differences between the two beers were in the mouthfeel and hop burn, with the Julius isolate "beating" WY1318 in both categories. I could not detect any real flavor differences, but the typical stone fruit notes were there in both. I have tried this recipe with Conan (Yeast Bay) and preferred WY1318 in a head to head there, fyi.

Combine these observations with the fact that it doesn't gunk up my fermentor/hop bags like WY1318 (damn you crop topper) and I'm definitely going to use it again.
 
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isomerization

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Will have a closer look at this tomorrow, but based on a quick glance it seems like your isolate is distinct from the other strains you've included and WY1056 is the closest relative. Interesting yes that the Vermont Ale and WY1968 seem closely related :)
Thanks! I appreciate your input greatly.

I have a new gel that I think provides (the potential for) more insight. I used two different primer pairs that have been reported in the literature, delta12-delta2 (left gel) and delta12-delta21 (right gel):



The strains are the same order as before with the addition of an 11th yeast (harvested from Tree House Double Shot).
 
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This is cool. I did biochem back in university, though I never worked in the field. Man, prepping an acrylamide gel was such a PITA back then. I hope it's easier these days.

Looking forward to more!
 

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Will have a closer look at this tomorrow, but based on a quick glance it seems like your isolate is distinct from the other strains you've included and WY1056 is the closest relative. Interesting yes that the Vermont Ale and WY1968 seem closely related :)
Unsurprised that Vermont Ale resembles WY1968 on the level of DNA sequence as the source material we used to isolate Vermont Ale from uses a strain that was rumored to originally have British origins. I'd say that's where the similarities start and end! WY1968 is a lower attenuator, high flocculator, produces decent amounts of diacetyl and has a fruity but restrained ester profile. Vermont Ale is a higher attenuator (typically around 80%), low flocculator, does not produce diacetyl in any appreciable quantities and has a massive ester profile of peaches/apricots.
 
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Unsurprised that Vermont Ale resembles WY1968 on the level of DNA sequence as the source material we used to isolate Vermont Ale from uses a strain that was rumored to originally have British origins. I'd say that's where the similarities start and end! WY1968 is a lower attenuator, high flocculator, produces decent amounts of diacetyl and has a fruity but restrained ester profile. Vermont Ale is a higher attenuator (typically around 80%), low flocculator, does not produce diacetyl in any appreciable quantities and has a massive ester profile of peaches/apricots.
Thanks for commenting! I do think it's a bit of an over generalization to just say both came from the U.K., so they're similar but the point is taken. I'd like to analyze other British strains if I can get my hands on some. It's still pretty interesting (to me at least) that they are so different phenotypically yet seem to share a lot of DNA similarity.

Since you'd be precisely to person to ask, why haven't we seen a commercially offered isolate from Tree House products? The yeast looks to be different than known NE IPA yeasts and performed favorably (n=1). I realize you may not want (or be allowed) to answer that, but I figured I'd ask anyways.
 

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Thanks for commenting! I do think it's a bit of an over generalization to just say both came from the U.K., so they're similar but the point is taken. I'd like to analyze other British strains if I can get my hands on some. It's still pretty interesting (to me at least) that they are so different phenotypically yet seem to share a lot of DNA similarity.



Since you'd be precisely to person to ask, why haven't we seen a commercially offered isolate from Tree House products? The yeast looks to be different than known NE IPA yeasts and performed favorably (n=1). I realize you may not want (or be allowed) to answer that, but I figured I'd ask anyways.

My supposition was that they may have branched off from a common lineage, more recently than not. Can't really speak to the isolation efforts of others, and I'm not saying we're isolating this strain, but we are currently in the process of isolating a new strain that is similar. Just posted about it on The Yeast Bay Facebook page:

"Everything is back in stock on the online store! Also, Brettanomyces bruxellensis - Strain TYB261 will be available to homebrewers shortly, and we'll likely be posting stock next week.

We also have yet another beta in the works that hasn't even been assigned a TYB number yet. We just streaked out the source material and are excited to get into characterizing the many isolates we'll pick. This house yeast, isolated from another brewery in the Northeastern United States, is similar to the Vermont Ale strain though the ester profile is less peach/apricot driven and more closely resembles citrus/pineapple/mango/guava. Keep your eyes peeled for updates, we're hoping to get through the isolation and characterization of this strain and make it available to commercial brewers and homebrew folks by the middle-end of May.

Cheers!"
 
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isomerization

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My supposition was that they may have branched off from a common lineage, more recently than not. Can't really speak to the isolation efforts of others, and I'm not saying we're isolating this strain, but we are currently in the process of isolating a new strain that is similar. Just posted about it on The Yeast Bay Facebook page:

"Everything is back in stock on the online store! Also, Brettanomyces bruxellensis - Strain TYB261 will be available to homebrewers shortly, and we'll likely be posting stock next week.

We also have yet another beta in the works that hasn't even been assigned a TYB number yet. We just streaked out the source material and are excited to get into characterizing the many isolates we'll pick. This house yeast, isolated from another brewery in the Northeastern United States, is similar to the Vermont Ale strain though the ester profile is less peach/apricot driven and more closely resembles citrus/pineapple/mango/guava. Keep your eyes peeled for updates, we're hoping to get through the isolation and characterization of this strain and make it available to commercial brewers and homebrew folks by the middle-end of May.

Cheers!"
Very cool! I look forward to picking up a vial.
 

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As always, I'm late to the party. I'm Bryan of Sui Generis Brewing, which has been cited here a little. Anyways, some replies to some previous posts...

Using a PCR method (https://suigenerisbrewing.blogspot....howComment=1490284392639#c8540474511060838192), I was able to amplify the ITS region (http://sites.biology.duke.edu/fungi/mycolab/primers.htm) of this yeast (my band is ~800 bp) ... I then sequenced the amplicon, and used BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to search for similar deposited sequences. I think this is where I've run into a wall, as the best hits I get back are only ~89% identical (they are S. cerevisiae).
With that said, I went back and looked at the DNA chromatogram a bit closer, and there is a suspicious amount of overlapping peaks.
89% homology is awfully low for an ITS sequence; I typically see 99-100% homology for yeast. Did you trim your sequence to remove poorly resolved bases (usually ID'd as an 'N' in the chromatagram/sequence file)? The mixed peaks may be a sign of contamination, but assuming there are two strains and both strains are S. cerevesea, it shouldn't matter as ITS sequences are pretty well conserved between species. Unfortunatly ITS size isn't a useful measure either, as pretty much any Eukaryote (including yourself) will produce an ~800bp ITS band. As Surgork already mentioned, you cannot do strain ID with ITS and need to reply on other methods. Inter-delta is the norm, but as you've already experienced, it needs to be compared to a database of other fingerprints to ID the strain.

Unsurprised that Vermont Ale resembles WY1968 on the level of DNA sequence as the source material we used to isolate Vermont Ale from uses a strain that was rumored to originally have British origins. I'd say that's where the similarities start and end! WY1968 is a lower attenuator, high flocculator, produces decent amounts of diacetyl and has a fruity but restrained ester profile. Vermont Ale is a higher attenuator (typically around 80%), low flocculator, does not produce diacetyl in any appreciable quantities and has a massive ester profile of peaches/apricots.
This is a good example of why even interDelta can be of only modest value. It doesn't take much to alter ester profiles or flocculation (and therefore attenuation) characteristics of yeast - there are many examples out there of single point mutations doing just that. interDelta can let you follow the parentage of yeasts, but some strain-specific changes can be "below detection" for even this method. Full genome sequencing is about the only way to get to that level of resolution, but the cost of that is still a bit beyond what is practical for the home brewer.

Just my $0.02

Bryan
 

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As always, I'm late to the party. I'm Bryan of Sui Generis Brewing, which has been cited here a little. Anyways, some replies to some previous posts...




89% homology is awfully low for an ITS sequence; I typically see 99-100% homology for yeast. Did you trim your sequence to remove poorly resolved bases (usually ID'd as an 'N' in the chromatagram/sequence file)? The mixed peaks may be a sign of contamination, but assuming there are two strains and both strains are S. cerevesea, it shouldn't matter as ITS sequences are pretty well conserved between species. Unfortunatly ITS size isn't a useful measure either, as pretty much any Eukaryote (including yourself) will produce an ~800bp ITS band. As Surgork already mentioned, you cannot do strain ID with ITS and need to reply on other methods. Inter-delta is the norm, but as you've already experienced, it needs to be compared to a database of other fingerprints to ID the strain.



This is a good example of why even interDelta can be of only modest value. It doesn't take much to alter ester profiles or flocculation (and therefore attenuation) characteristics of yeast - there are many examples out there of single point mutations doing just that. interDelta can let you follow the parentage of yeasts, but some strain-specific changes can be "below detection" for even this method. Full genome sequencing is about the only way to get to that level of resolution, but the cost of that is still a bit beyond what is practical for the home brewer.

Just my $0.02

Bryan
Well said Bryan, this is useful information that's worth far more than $0.02! :mug:
 
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Regarding the ITS sequencing, I think it was a problem on the sequencing end, rather than a true heterogenous population. But, as many have pointed out on this thread now, ITS sequencing wasn't going to be helpful in this situation anyways. So I dropped it and switched to interdelta PCR, which I was not aware of until I started playing around with this strain.

Personally, I think interdelta PCR was very helpful here not because it revealed shared parental lineage, but because it didn't! It appears to suggest that the yeast isolated from a Tree House can is different than any of the other traditionally used (by homebrewers at least) NE IPA yeasts. I think that's pretty cool!

Phenotypic characterization is more important for us homebrewers anyways, but I think its still cool to ask and try to answer these types of questions.
 

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I forgot to add that the Julius isolate is certainly unique via this DNA fingerprinting method!

So, what this really means is that I need more yeast samples to analyze, I'm looking into figuring out an easy way to mail samples through the postal service (only costs a stamp!), if anyone has suggestions and is interested in sending yeast that is not on my current image, please let me know!!
I'd really like to see how Fermentis S-04 and S-05 compare. If you want to PM me your address, I'll just order a pack of each and have them shipped to you.
 
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I'd really like to see how Fermentis S-04 and S-05 compare. If you want to PM me your address, I'll just order a pack of each and have them shipped to you.
PM sent. I also picked up T-58, S-23, W-34/70 and Danstar Munich dry yeasts to look at as well ($6 including shipping for all 4 since they're old).
 

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As always, I'm late to the party. I'm Bryan of Sui Generis Brewing, which has been cited here a little. Anyways, some replies to some previous posts...




89% homology is awfully low for an ITS sequence; I typically see 99-100% homology for yeast. Did you trim your sequence to remove poorly resolved bases (usually ID'd as an 'N' in the chromatagram/sequence file)? The mixed peaks may be a sign of contamination, but assuming there are two strains and both strains are S. cerevesea, it shouldn't matter as ITS sequences are pretty well conserved between species. Unfortunatly ITS size isn't a useful measure either, as pretty much any Eukaryote (including yourself) will produce an ~800bp ITS band. As Surgork already mentioned, you cannot do strain ID with ITS and need to reply on other methods. Inter-delta is the norm, but as you've already experienced, it needs to be compared to a database of other fingerprints to ID the strain.



This is a good example of why even interDelta can be of only modest value. It doesn't take much to alter ester profiles or flocculation (and therefore attenuation) characteristics of yeast - there are many examples out there of single point mutations doing just that. interDelta can let you follow the parentage of yeasts, but some strain-specific changes can be "below detection" for even this method. Full genome sequencing is about the only way to get to that level of resolution, but the cost of that is still a bit beyond what is practical for the home brewer.

Just my $0.02

Bryan
Single point mutations can be huge, just look at teosinte vs corn, it only took like four mutations, but you wouldn't recognize thm as the same species. Same for grapes, people did some sequencing and it turns out many of the French grapes are literally siblings.

I think it helps though to know when you have essentially the same strain producing different flavors vs a strain that is close to a different species.
 

trav77

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Single point mutations can be huge, just look at teosinte vs corn, it only took like four mutations, but you wouldn't recognize thm as the same species. Same for grapes, people did some sequencing and it turns out many of the French grapes are literally siblings.

I think it helps though to know when you have essentially the same strain producing different flavors vs a strain that is close to a different species.
It took only a small number of mutations to get rid of the rock-hard glume in teosinte and make it edible. After that there was an incredible amount of de novo variation and selection by humans to get to what is now corn.

But yes, a single point mutation can be huge in certain cases.
 

trav77

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Kind of late to the game here but have you looked into doing a genome wide SNP fingerprint? Pretty cheap these days. I haven't done much work with yeast but it's a model organism so no doubt there are vast resources out there to do this type of work. That would let you do a phylogenetic analysis and would give a better representation of relatedness than a handful of loci via PCR. Just my 2c. Cool project!
 

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