As previously communicated in my last post, I am currently working on a Dekkera anomala proteome (strain YV396) to get a better understanding of the pathways associated with compounds found in beers like Lambics. I compiled my data and first results into a easier to read format (see below). In case there is something else of interest in the genome, I will likely publish these results on my blog as well.
It has been a while since I look at Brettanomyces genomes since there wasn’t that much data available to play around. And mainly B. bruxellensis data is available due to its importance in the wine industry. This all changed when I came across the deposited draft genome assembly of a Dekkera anomalus YV396 genome in June by Vervoort,Y et al. Since there was no annotation material available for this genome I quickly decided to give the annotation a shot myself. Simply because I am interested in certain pathways in Brettanomyces. Everything I needed was my Ubuntu notebook (which died during the annotation process), my new Ubuntu workstation (replacing the notebook) and some Python coding. No access to a cluster whatsoever. Is it possible to finish an entire annotation project at home? You will find out very shortly. As I am still compiling data for a post, I want to start sharing the material part as well as the first abstract. Just to give you a sneak-peek into the project. The remaining part of the genome & proteome project will get published very soon. Just give me some additional time to finish up the various pathway analysis and writing up the paper. Still a lot to be discovered in the new genome…
I – Methods
The draft genome assembly of Dekkera anomalus strain YV396 (isolated from a Belgian brewery) was retrieved from GenBank (accession number LCTY00000000.1; June 2015) deposited in May 2015 by KU Leuven [Vervoort et al.]. Illumina HiSeq data (100x coverage) was assembled into a genome using SOAPdenovo v.1.05. The statistics for the obtained assembly are summarized in Tab. 1.
Gene prediction on contigs was performed using the AUGUSTUS web-service (AUGUSTUS parameter project identifier: pichia_stipitis, UTR prediction: false, report genes on both strands, alternative transcripts few, allowed gene structure: predict any number of (possibly partial) genes, ignore conflicts with other strand: false) [Stanke et al.2006, 2008]. The gene prediction statistics are summarized in Tab. 2.
Gene annotation was performed by Blast2GO including remote blastx on NCBI and InterProScan for domain predictions [Conesa et al, 2005]. GO-term mapping and annotation performed by Blast2GO pipeline. Close to 3,000 out of the predicted 4,160 could be annotated by Blast2GO (Fig 3). Another subset of about 600 sequences could be mapped to a biological function without a GO term and about 460 sequences only resulted in BLAST hits which could not be further associated with a protein function.
Most abundant species associated with the best blastx hits were Dekkera bruxellensis, Ogataea polymorpha and Pichia kudriavzevi (not shown).
- Conesa, A., Götz, S., García-Gómez, J. M., Terol, J., Talón, M., and Robles, M. (2005). Blast2GO:a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics, 21(18):3674–3676.
- Stanke, M., Diekhans, M., Baertsch, R., and Haussler, D. (2008). Using native and syntenically mapped cDNA alignments to improve de novo gene finding. Bioinformatics, 24(5):637–644.
- Stanke, M., Schoffmann, O., Morgenstern, B., and Waack, S. (2006). Gene prediction in eukaryotes with a generalized hidden Markov model that uses hints from external sources. BMC Bioinformatics, 7(1):62.
- Vervoort,Y., Herrera-Malaver,B., Mertens,S., Guadalupe Medina,V., Duitama,J., Michiels,L., Derdelinckx,G., Voordeckers,K., Verstrepen,K.J. (2015) Purification and characterization of a novel Brettanomyces anomalus beta-glucosidase enzyme suitable for food bioflavoring – unpublished.
Starting 2015 with science. This is a more in depth post about yeast phylogeny. Please have a look at the more basic post here. This time, I would like to discuss the relationship of various Brettanomyces/Dekkera strains and share my results concerning the recent WLP Brettanomyces bruxellensis Trois yeast ID crisis.
I would like to start with the Brettanomyces/Dekkera strains first. I obtained 38 Dekkera/Brettanomyces sequences (26S rDNA) from the CBS database, aligned them using MUSCLE (run in default mode, see MSA in Fig 1) and reconstructed a phylogenetic tree using MABL (model HKY85) and rendered using TreeDyn (run in default mode, Fig 2).
I would like to emphasize the importance of having a look at the intermediate steps in constructing a phylogenetic tree since sequence alignments can happen in various ways. Looking at the MUSCLE output, one can already expect to see the individual strains clustered together due to their sequence similarities. However, there is one particular sequence that seems to be a bit off (sequence number 1, B. naardenensis CBS 6116). This sequence seems to be different from all the other B. naardenensis sequences shown at the bottom (sequences 29-38). Based on this result, one can expect to see CBS 6116 to be an out-group to the other B. naardenensis sequences. In summary, the alignments seem to be okay and let’s have a look at the phylogenetic tree of the 38 sequences.
As already observed in the MSA, the individual Brettanomyces/Dekkera species cluster together as expected (Fig 2). B. naardenensis CBS6116 does indeed form an out-group (isolated from lemon drink in France) and is kind of distant to the other B. naardenesis sequences. So why is this CBS 6116 sequence different?
To address this question, I tried to figure out first what other sequences are similar to CBS 6116 by BLASTing against the NCBI nr database (run in default mode). I got several hits and manually inspected the results via alignment. The CBS 6116 sequence has high sequence identities to Pichia guillermondii (Fig 3). One might reason – based on these results – that CBS 6116 is not a Dekkera/Brettanomyces strain.
Going back to the phylogenetic tree in Fig 2. It seems that all the current Dekkera/Brettanomyces species end up in the same clusters and show enough substitutions to tell individual species apart. This is very important information for everyone interested in determining the species of an unknown Dekkera/Brettanomyces strain.
WLP Brettanomyces Trois ID crisis
I got interested in the WLP story (WLP Brettanomyces bruxellensis Trois not being a Brettanoymces strain) and started by looking at the WLP Brett Trois ITS sequences I received from Omega Labs (reads 64-ITS1.ab1 and 64-ITS4.ab1). I had a quick look at the chromatograms and would not use any of the reads for my own work due to its low base quality and multiple peak calls at certain positions. Besides the read from Omega Labs, I received a read used to ID the B. Trois as Saccharoymces cerevisiae at Charles River’s Lab as well as the read published by SuiGeneris. Since I don’t have the chromatograms for these two sequences, I manually inspected the reads in various alignments to get an idea about their quality. Lets start with my analysis for the WLP Brettanoymces bruxellensis Trois ID’ing based on phylogenetic trees.
To get the most likely phylogenetic tree one has to follow some basic rules. Beginning with looking at the same shared derived homologous traits (homologous DNA sequences) and verifying that no other DNA sequence alterations impact the phylogenetic tree like sequencing errors (wrong base calls, no base call, multiple base calls etc). So far so good. I aligned some ITS2 regions from various CBS Saccharomyces strains to the various ITS2 reads from WLP’s Brettanomyces Trois (Fig 4).
Lets have a look at the alignment shown in Fig 4. Especially at the 64-ITS4.ab1 read from WLP B. Trois (sequence 1 shown at the top). One can easily see, that various base differences exists compared to the other sequences in the alignment. Supporting the initial idea that the DNA sequence from this read is not very reliable nor very representative (if one compares the read to the two other WLP B. Trois reads shown at the bottom). Due to the differences in this read, I would not be surprised to see this read out-grouped in the phylogeny tree later on. The two reads from Charles River’s Lab and SuiGeneris seem to be way better and similar to other Saccharomyces sp. sequences shown in the alignment.
To make the phylogeny more computational efficient and more reliable, I extracted the sequences mapping to the two WLP sequences (sequence regions from the right side in Fig 4) to receive the alignment shown in the next figure (Fig 5).
A first look at the alignment in Fig 5 reveals some hotspots for variations like gaps and different base calls (color regions). The question I would like to address now is whether the variations are due to speciation or artifacts. Artifacts are commonly more random than variations due to speciation. A quick inspection reveals lots of random variations in the 64-ITS4 read but none/few for the two other WLP B. Trois reads.
The phylogeny tree obtained for the sequences shown in the alignment in Fig 5 is shown in Fig 6
As expected the n64-ITS4 read gets out-grouped and might be interpreted as a different species. Well this is true if one just looks at the tree in Fig 6 but did not look at the previous alignments and possible reasons for the out-grouping. In this case, the quality of the base calls for the n64-ITS4.ab1 read are very poor and led to wrong base calls after all (investigated by pair-pair alignments). In summary, the differences leading to an out-grouping of WLP B. Trois based on the n64-ITS2 read are not due to the physical differences in the DNA sequence but due to sequencing errors.
In summary, the two WLP B. Trois sequences group together with other Saccharomyces cerevisiae strains. Supporting the current view that WLP B. Trois might indeed not be a Dekkera/Brettanomyces strain (at least based on ITS2 sequence homology). Or at least the samples of WLP B. Trois that float around these days.
As I do isolate and work with my own Brettanomyces strains, it happened to me various times that I was able to observe some Saccharomyces cerevisiae beside the initial Brettanomyces strain after some serial re-pitches (and I did not use Saccharomyces for the fermentation). This Saccharomyces contamination might lead to problems during the propagation. Saccharomyces cerevisiae might overgrow Brettanomyces increasing the Saccharomyces:Brettanomyces ratio even further. Eventually leading to very few Brettanomyces cells left in a population. Keeping Brettanomyces samples is not as easy as one might think. And I would not be surprised if more yeast conundrums turn up in the next years.
Eureka, we are back to Brett experiment updates. I would like to take the opportunity here to remind people to fill in evaluation data they have so far. At this point, only 6 people (out of 38 collaborators) sent in data either via the provided google doc form or by posting their results on blogs. If you haven’t sent in any data so far, may I kindly ask you to do so? Sending me your evaluation sheets as PDF works as well.
That’s it already. More yeast posts to come soon.
Hello everyone. This is the fourth update concerning the BBA/EBY Brett Experiment and should give you some preliminary results for some of the strains and be a reminder concerning the evaluation process. I hope the experiment is working fine so far. As mentioned in the last update (BBA/EBY Brett Experiment Update 3) the planned time points for the evaluation are
- Month 1 (uncarbonated at bottling), Month 2, Month 6, 1 Year
and the results should be put into the following list
- Google Form of the Brett sheet set up by Luke and adapted by Jeff
Whenever possible, please transfer all your data from whatever form you used to the Google Form mentioned above. If you include any other (commercial) Brett strains in the experiment, please use the same form and just mention the strain in the appropriate field (yeast code).
As far as I know, two bloggers published first results so far.
- http://comparebeer.blogspot.com/2013/11/blog-post.html (at bottling)
If someone else published evaluation data on their blog (or wherever) and is not mentioned here, please let me know. For the Google Form above, only one person filled in information so far. A bit too early to make any conclusions so far
Small lab update
I would like to end with a short update about the current status of the EBY lab. I am still isolating new yeasts from various new beers (mostly from the US now) and am now especially interested to get some Lactobacillus strains as well. And I finally detected some living bacteria in a dreg sample (see picture below) which might be Lactobacillus (will not reveal the brewery/beer at this point). On the other hand, I got some non-Saccharomyces yeasts from various dregs as well. One might be especially interesting as it is from a brewery from the UK isolated from a barrel aged beer. And I am quite sure they did not add Brettanomyces artificially. In addition, I finally got my hands on a Berliner Weisse brewed with an old Brettanomyces strain isolated from a very old, traditional Berliner Weisse. Will see if I can manage to get some living yeast from the bottle though. Further on, I am playing around blending various strains and dregs to get some unique and aggressive blends. That’s it so far.
On yet another unrelated notice, I would be interested using the legendary Conan yeast for a future batch. However, it is impossible to get the beer in Europe to isolate the yeast myself nor any yeast shipped to Europe. If anyone out there willing to send me some Conan, please write an email. Thanks for reading and stay tuned
Hello everyone. This is the third update concerning the BBA/EBY Brett Experiment and should give you further information about the evaluation of the beers and yeast strains. After some issues with leaking tubes, it all seems to be fixed now. At least I did not receive any feedback so far about problems with any of the yeast strains. I learned one lesson already which is to use a different kind of tube to send out yeasts in the future.
If you are interested in any upcoming strain releases and general news about my yeast lab, please go to https://groups.google.com/forum/#%21forum/eureka-brewing-yeast
I would like to thank all the people for sending me the money for the shipping and am especially thankful for all the ones who sent me some extra as well. This money will flow back into the lab and any yeast related projects. Thank you for that. Unfortunately, there are still three people who did not send me the money for their shipping yet. I might have to cover these shipping costs myself as it seems.
And I got my first yeast package as well. My thanks to Barrett for sending me some dregs of Jester King’s Das Wunderkind and Green Flash’s Rayon Vert. Already excited to have a closer look at these dregs. And since I am already talking about dregs, I recently put aside the dregs of Lost Abbey’s Amorosa de Framboise and Mo Betta Bretta. Again, really excited to see what I can get out of these dregs. I already have enough material for a second round of Brett Experiment…
Sensory evaluation and the Brett score sheet
Jeff put together a pretty nice Brett score sheet in the beginning which was transformed into a Google Form by Luke later on. There is nothing wrong in using Jeff’s Brett sheet for the evaluation process as I prefer to do my tastings with a pencil and paper. The Google Form includes the same structure as Jeff’s form but makes it a bit easier for us to do the evaluation a bit easier afterwards. Whenever possible, please transfer all your data from the Brett sheet to the Google Form. We ask some background information in the Google Form to have additional information available for the evaluation process later on. Here are the links to the mentioned score sheets:
- Jeff’s Brett sheet:
- Or a direct link: https://docs.google.com/file/d/0B3kKViCCOyVmbF9WT0ROYnR5dXc/edit?usp=sharing
- Google Form of the Brett sheet set up by Luke and adapted by Jeff
If you include any other (commercial) Brett strains in the experiment, please use the same form and just mention the strain in the appropriate field (yeast code).
When do I sample the beers?
The planned tasting time points are as following:
- Month 1 (uncarbonated at bottling), Month 2, Month 6, 1 Year
Just mention the closest time point if you for example taste the beers after seven months instead (like 6 months for this example).
How/where to sample the beers?
As Ryan pointed out, to get more evaluation data, it is a good idea to share the beers with other homebrewers, professional brewers, beer judges, beer geeks, friends etc. Either by sitting together to do the actual tasting or sharing bottles with others. How you do it is up to you and it is not mandatory to trade nor share the beers with others. It’s just an idea. Jeff for example is thinking of offering some spots for a tasting panel in the San Diego area (http://jeffreycrane.blogspot.com). On my side (although I haven’t actually brewed the batch yet) will share the beers with a recently set up Wild Ale/extreme beer geek group in Switzerland at one of our upcoming meetings. If you are from/living in Switzerland and are interested in either our newly formed group or be present at the tasting of the beers, please write me an email (firstname.lastname@example.org) and we will figure out things.
The question here is how to do the organization. As I do not want to give away any collaborator’s email address without even asking, we could use the HBT forum thread about this experiment to organize swaps and meetings. If you have better ideas how to do the organization, please let me know.
I would like to finish with a few links to other blogs writing about this experiment. Please let me know if I forgot someone.
Hello fellow BBA/EBY experiment collaborators. This is the second update concerning the BBA/EBY Brett Experiment. The first update can be found here. Once again thanks to all the participants and all the people offering to send me some unique dregs and yeasts as well. I would like to proceed with some numbers:
- 36 collaborators are officially in for the experiment (me not included)
- 347 samples will be sent out for the experiment
- 9 collaborators will test the entire 20 strains (awesome!)
- From the 36 collaborators, 1 is not from the US (and it isn’t me). Don’t worry, I only looked up the cities and not the entire addresses 🙂
I am sorry to announce that I don’t accept any further collaborators. If you are interested in the strains, please subscribe to https://groups.google.com/forum/#!forum/eureka-brewing-yeast to get email alerts of future strain releases
As previously mentioned, the yeasts will be sent out on Monday, the 26th of August. Please read the first update post what has to be done after you received the yeasts.
First results about the strains
I would like to publish first results about some of the strains
- EBY001 B. girardin I: The strain which will be released is different from the one I originally isolated. The original strain looked and behaved like normal brewers yeasts and I therefore replaced it with another Girardin Brettanomyces strain I have
- EBY005 B. cantillon I, EBY008 B. cantillon II, EBY009 B. cantillon III, EBY011 B. cantillon V, EBY013 B. cantillon VII and EBY016 B. lembeek I are really slow growers. Expect to wait longer to get signs of fermentation
- All strains can form colonies on agar plates. Therefore all strains are viable, even the slow-growing ones