tag:blogger.com,1999:blog-8081751738815878503.post116549060911453798..comments2023-04-14T03:40:29.342-04:00Comments on Sui Generis Brewing: Yeast Identification TestBryanhttp://www.blogger.com/profile/16672407110077541595noreply@blogger.comBlogger16125tag:blogger.com,1999:blog-8081751738815878503.post-70625672468268734602016-03-23T12:30:12.767-04:002016-03-23T12:30:12.767-04:00It is impossible to say, as the morphology of Bret...It is impossible to say, as the morphology of Brett can range from Sacc-like ovoids through to hyphae-like branching yeasts. You could ferment with it to see if you get the expected flavour profile; if straight from white labs it is unlikely to be seriously contaminated.Bryanhttps://www.blogger.com/profile/16672407110077541595noreply@blogger.comtag:blogger.com,1999:blog-8081751738815878503.post-175864771411275102016-03-23T12:03:01.190-04:002016-03-23T12:03:01.190-04:00Sorry to resurrect this thread but looking for you...Sorry to resurrect this thread but looking for your input on a observation that I made: microscopic examination of WLP653 revealed that the cells exhibited a Saccharomyces morphology instead of the Brett morphology of WLP648 and WLP650. I don't know whether I have a contaminated vial of WLP653 or whether this is indeed what the strain is supposed to look like. Although PCR might answer the question, I don't want to go to the expense of ordering primers. Look forward to a response.<br /><br />Cheers<br /><br />OlaAnonymoushttps://www.blogger.com/profile/13353234511827896871noreply@blogger.comtag:blogger.com,1999:blog-8081751738815878503.post-83058647277150183492013-05-12T21:35:05.984-04:002013-05-12T21:35:05.984-04:00Sam, it looks like I have to eat crow*. The sequen...Sam, it looks like I have to eat crow*. The sequences arrived on a Sunday (imagine that). There was huge 26S variability between the bruxellensis and lambicus strains. I have the post up for these sequences, so look for the details there:<br /><br />http://suigenerisbrewing.blogspot.ca/2013/05/identifying-wild-yeast-part-deux.html<br /><br />Bryan<br /><br />*In the real world I have eaten crow - it was rather good, so I don't really understand this saying....Bryanhttps://www.blogger.com/profile/16672407110077541595noreply@blogger.comtag:blogger.com,1999:blog-8081751738815878503.post-4444722372935261952013-05-12T10:58:46.052-04:002013-05-12T10:58:46.052-04:00I only have access to a partial copy of the 4th ad...I only have access to a partial copy of the 4th addition at the moment, via google books; my access to the 5th edition is limted to when I'm at work. I'm using the 4th version for my upcoming blog post as I can link to the relevant page:<br />http://books.google.ca/books?id=bnWQsv2eTcEC&pg=PA51#v=onepage&q&f=false<br /><br />That said, I'm 99% sure the same table appears in the 5th edition...somewhere in the first 1/3rd of the book if I recall.<br /><br />Short version is they used eletrophoretic mobility (a method common pre-modern genetics) to measure evolutionary differences in a few enzymes. Using this method, "b. lambicus" appears as two distinct populations; one entirely within brux, and one mid-way between brux & anomalus.<br /><br />I've been collecting some of the 26S rRNA sequences from the b. lamb/brux/anomala strains mentioned in the table in "Yeasts"; alignments of those appear to mimic what was reported in "yeasts" - some lambichs strains (e.g. CBS 75, ) are 99.5% to 100% identical to the proteotypical brux; while one (CBS 5602) is clearly an outlier and actually clusters closer to the one anomala sequence I currently have.<br /><br />BryanBryanhttps://www.blogger.com/profile/16672407110077541595noreply@blogger.comtag:blogger.com,1999:blog-8081751738815878503.post-3197401338874244382013-05-12T08:42:41.624-04:002013-05-12T08:42:41.624-04:00Can you tell me on what page in "Yeasts - A T...Can you tell me on what page in "Yeasts - A Taxonomic Study" by Kurtzman et al (I have the 5th edition) they show the discrete Lambicus clades? I can't find it. On p. 375 (Chapter 25) they list B. lambicus as a synonym for the D. bruxellensis species. Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-8081751738815878503.post-5015116727318560882013-05-11T14:32:50.750-04:002013-05-11T14:32:50.750-04:00I agree that there may be differences, but even in...I agree that there may be differences, but even in the variable regions, rRNA evolves slower than other genomic regions. I should have the sequences early next week, so we'll have a better idea then.<br /><br />As for B. lamb, its taxonomical status is uncertain. The ICBN oversees fungal plant species nomenclature; lambicus remains an accepted species and strain identifier in their catalogue (which is odd, as it should be one or the other, never both). In 'The Yeasts - A Taxonomic Study', the review on brettanomyces/dekkara shows that lambicus forms two discrete clades within the broader bruxellensis group; one inseparable from the prototype brux, and one mid-way between brux and anomala (about 1% variation).<br /><br />Long story short, in the real world there are not clear divisions between species. What we call lambicus is actually two unique clades; one an inseparable part of the brux group of species, the other an outlier mid-way between prototypical brux and prototypical anomala.<br /><br />As for my B. lambicus strain, I have no idea which "group" of lambicus it belongs to. I've already got the B. brux ad B. anomala rRNA sequences from the strains used in "The Yeasts"; a 3-way comparison should show which lambicus I have - assuming it fits within the 2-clade paradigm...Bryanhttps://www.blogger.com/profile/16672407110077541595noreply@blogger.comtag:blogger.com,1999:blog-8081751738815878503.post-59164997069589556792013-05-11T13:59:54.040-04:002013-05-11T13:59:54.040-04:00From a taxonomy-genetic point of view, B. lambicus...From a taxonomy-genetic point of view, B. lambicus and B. bruxellensis might differ in their rDNA sequences by less than 0.5%. In other words, less than 3 nucleotides in the D1/D2 LSU rDNA. Sorry to correct you there, B.lambicus is a synonym of B. bruxellensis. Not a subspecies. <br /><br />Cheers, SamAnonymousnoreply@blogger.comtag:blogger.com,1999:blog-8081751738815878503.post-17205948890717107522013-05-09T16:36:13.205-04:002013-05-09T16:36:13.205-04:00The PCR didn't work out as well as I hoped, wi...The PCR didn't work out as well as I hoped, with only the B. bruxellensis and B. lambicus PCRs working (kinda what like happened here - I'm thinking my DNA isolation on the later 2 strains was lacking). I dropped the DNA off for sequencing today, so I should have the answer early next week.<br /><br />My guess is there may be a change or two, but I'm not expecting much. Technically, B. lambicus is actually a subspecies of B. bruxellensis. So differences may be small.<br /><br />BryanBryanhttps://www.blogger.com/profile/16672407110077541595noreply@blogger.comtag:blogger.com,1999:blog-8081751738815878503.post-91848325258057719202013-05-09T11:45:18.824-04:002013-05-09T11:45:18.824-04:00You are welcome. I would be interested to know if ...You are welcome. I would be interested to know if you pick up sequence differences between B. bruxellensis and B. lambicus?<br /><br />Cheers and good luck with your next experiments, <br />SamAnonymousnoreply@blogger.comtag:blogger.com,1999:blog-8081751738815878503.post-7869581572396996362013-05-06T08:54:56.878-04:002013-05-06T08:54:56.878-04:00Sam, I'd just like to thank you again for thos...Sam, I'd just like to thank you again for those papers. Two covered 26s ribosomal sequencing, and had great success with the NL-1 and NL-4 primers - and they sequenced the very species I'm likely to find (Sacch, Brett, Hanisporia, etc). I've ordered those primers and will hopefully be able to post again soon using the new ones.<br /><br />I just thought I comment on the DNA fingerprinting - that is a pre-(cheap) sequencing method that relied on using the size of various PCR or digest fragments to ID species. While it works well, you need to have a pre-existing database of fragment sizes. Unfortunately, those databases are generally incomplete, as cheap sequencing displaced them in the late 1990's.<br /><br />Again, thanx for the papers!<br /><br />BryanBryanhttps://www.blogger.com/profile/16672407110077541595noreply@blogger.comtag:blogger.com,1999:blog-8081751738815878503.post-47732663063728595732013-05-03T14:43:48.398-04:002013-05-03T14:43:48.398-04:00Thanx! I'd only read about 1/2 of those, so I...Thanx! I'd only read about 1/2 of those, so I've got some more reading to do.Bryanhttps://www.blogger.com/profile/16672407110077541595noreply@blogger.comtag:blogger.com,1999:blog-8081751738815878503.post-19753359297705757132013-05-03T13:55:15.250-04:002013-05-03T13:55:15.250-04:00Below some publications about yeast PCR methods I ...Below some publications about yeast PCR methods I read so far. Maybe a multiplex PCR might work as well.<br /><br />- Cocolin et al (Molecular Detection and Identification of Brettanomyces/Dekkera bruxellensis and Brettanomyces/Dekkera anomalus in Spoiled Wines)<br /><br />- Curtin et al (Genetic diversity of Dekkera bruxellensis yeasts isolated from Australian wineries)<br /><br /> - Ibeas et al (Detection of Dekkera-Brettanomyces Strains in Sherry by a Nested PCR Method)<br /><br />- Boekhout et al (Phylogeny of the Yeast Genera Hanseniaspora (Anamorph Kloeckera) Dekkera (Anamorph Brettanomyces), and Eeniellaa as Inferred from Partial 26s Ribosomal DNA Nucleotide Sequences)<br /><br />- Hayashi et al (Detection and identification of Brettanomyces/Dekkera sp. yeasts with a loop-mediated isothermal amplification method)<br /><br />- Kurtzman et al (Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences)<br /><br />- Mitrakul et al (Discrimination of Brettanomyces ⁄<br />Dekkera yeast isolates from wine by using various DNA fingerprinting methods)<br /><br />Cheers, SamAnonymousnoreply@blogger.comtag:blogger.com,1999:blog-8081751738815878503.post-51366006706647844362013-05-03T08:49:43.836-04:002013-05-03T08:49:43.836-04:00The problem with 2 primers is it costs 2X as much ...The problem with 2 primers is it costs 2X as much - plus the ITS is only 300-500bp long, so you can get all of it (minus the bit by the primer) in one read. Like I said, my primer selection was a little naive; while some groups had great success, others didn't. I'm thinking seriously about the large subunit now - it looks to be as good, there are more sequences deposited covering a broader swath of yeasts, and its bigger so it should be possible to get firmer matches.<br /><br />I am aware of primer-BLAST, but I'm not a fan of it. Its not very good ad handling degeneracy in mixed samples. I have some matlab scripts which do a better job, assuming I feed it a reasonable set of genomes.Bryanhttps://www.blogger.com/profile/16672407110077541595noreply@blogger.comtag:blogger.com,1999:blog-8081751738815878503.post-36356181938680015482013-05-02T17:29:03.179-04:002013-05-02T17:29:03.179-04:00Thanks for your replies.
For the missing end. Thi...Thanks for your replies. <br />For the missing end. This makes sense if you only sequence from one side using one primer only. I am used to sequence stuff with two primers. In this case, I would have sent in the DNA with the forward and once with the reverse primer. Then I would get the sequences of both ends plus two sequencing results. <br /><br />Believe me, I read through a bunch of papers concerning yeast differentiation based on ITSs over the last years and I still haven't found a good way to apply ITS sequencing on our particular problem. That's why I am very sceptical about all ITS-based sequencing stuff. If one wants to stick to the ITS PCRs, multiple primers might be useful. Some specific for Saccharomyces, some specific for some Dekkera species and others for yeast species more abundant in beer such as Kloeckera, Pichia etc. That's why I might give the MALDI-TOF a go. Simply because it is easy and fast. The only disadvantage are the missing reference databases. <br /><br />I guess you are aware of primer-BLAST at NCBI. This gives you an idea what you might amplify using the primers you now have on hand. And there is another yeast hunter doing some work on yeast identifications: http://jaapie.org/?page_id=497 <br /><br />Good luck and I hope you get some great results soon, Cheers SamAnonymousnoreply@blogger.comtag:blogger.com,1999:blog-8081751738815878503.post-85094189246951481622013-05-02T16:31:20.254-04:002013-05-02T16:31:20.254-04:00One Q at a time:
I made some copying errors in mov...One Q at a time:<br />I made some copying errors in moving sequences over from the sequence files; there are three reads of Bret B sequence in there, instead of the consensus read of each. I'll try to remember to enter the correct ones tomorrow at work.<br /><br />The "missing" 5' sequence occurres because I use the same forward primer to sequence as I do to amplify out the genes. Because of the way sequencing works you tend to not get sequence closest to the primer, and instead get sequence ~50bp downstream of the primer.<br /><br />The 'extra' length of the one sequence is simply a product of the inaccuracy of gels for determining size. There are better gels with higher resolution (polyacrilimide gels), but for this kind of work we use the poor-quality gel (agarose) as they are easier to recover the DNA from. The caveat then becomes, especially for smaller pieces of DNA, that the gel only reflects the size to within ~100bp.<br /><br />As for the sequencing errors, it wouldn't be the enzyme; the additions of mutations are random, and the mutation rate low enough, that its statistically impossible to generate these sorts of read errors. The error I showed in the diagram was unusual; most of the errors are later in the sequence, where the peak-heights are small. These end-run errors are common in sequencing, and usually reflect nothing more than poor signal-to-noise in the machine.<br /><br />I'm not overly happy with the results - further reading (of course, after ordering the primers) led me to learn that the ITS region is best for other groups of fungi, but is less ideal for yeasts. Based on those readings I'm re-orientating my plan and instead will (probably) switch to large ribosomal subunit sequencing. But before then I'm going to download some genomes and do some "in silico" tests to make sure the new sequencing approach will work better. I.E. instead of just jumping in, I'll do all my homework first.Bryanhttps://www.blogger.com/profile/16672407110077541595noreply@blogger.comtag:blogger.com,1999:blog-8081751738815878503.post-1560833581451888322013-05-02T15:59:57.671-04:002013-05-02T15:59:57.671-04:00Hi Bryan,
nice write-up. I have some questions. I...Hi Bryan, <br />nice write-up. I have some questions. I did a multiple sequence alignment of your three sequences and the only differences between them are from the sequencing errors. Since this is the case, I see no point in allocating the sequences to different organisms if they show such high similarities. I further aligned the sequences to a rRNA sequence of D. bruxellensis (AM850055) and there are significant differences between the AM850055 sequence and yours. I therefore wouldn't conclude that some of your sequences map to Dekkera and others don't. For example, if you blast the 1084 sequence against the Dekkera database, you get Dekkera hits as well... However, with gaps like in the case of blasting WLP B. lambicus against the Dekkera database. <br /><br />Another question concerning the primers. Did you trim your sequences somehow? Because I can only find binding regions for the reverse primers. <br /><br />Yet another question. Why is the sequenced sequence of WLP B. lambicus (584 bp) longer than the one you see on the gel? <br /><br />As a side note, I did some blasting and for me its seems that you cannot for example amplify Kloeckera rRNA with your primers. Bokulich et al did not use these primers to amplify Kloeckera anyway. I therefore expect that several yeast cannot be identified with you actual setup. Maybe the reason why you couldn't amplify the mystery yeast. <br /><br />My last thoughts are about the sequencing errors. In my experience, sequencing errors most often occur in highly repetitive sequences. But in case of your sequences, the errors are not in repetitive sequences. Might this be because of the Taq polymerase you use? We use proof reading enzymes in our lab in case we want to sequence something. And the sequences we get are very low in sequencing errors. <br /><br />Good luck with your next experiments, cheers SamAnonymousnoreply@blogger.com