Tag Archives: sequencing

Basidiomycete genomes galore


Just finished attending Genetics and Cell Biology of Basidiomycetes in Cape Girardeau, MO which was an intimate gathering of basidiomycetaphiles.  I learned about systems that are used for studying fruiting body development, genetic mapping, pheromone and mating genes, kinesin dynamics, meoitic gene regulation, and a host of topics.  I’m happy I got a chance to meet more folks in the community and learned about where informatics and computational approaches are really needed to push along some of the interpretation of the more than a dozen basidiomycete genomes.  In particular it sounds like the PleurotusSchizophyllum, Agaricus bisporus, and Serpula genomes are all marching along to completion with some already in 4X assembly or further.  

GCBBVI Group Picture

So we’ll further have more samples from of key model and some less-model species to assist researchers working on many different mushroom-forming fungi that range from brown and white-rotting saprophyte fungi to mycorrhizal fungi that associate with plants.    I’m excited about the work to make transformation and knockouts more readily in these systems too to push the genetics and cellular biology of these systems even further.  The genome sequences will be another tool in these endeavors.

The last day ended with a discussion about genome annotation and future support for curating gene models.  Basically everyone is unhappy with computational predictions and want to be able to go in and fix things. (I think people remember the ones that are gotten wrong more readily than the ones that were right, but computational prediction definitely performs poorly in some situations).   In this Web 2.0-land we live in, this is still not something easily done with any of the freely available genome browsing tools. The JGI’s browser was lauded for its ability to handle these kinds of requests, but how do we proceed when genomes are not sequenced by that center or when (not too distant future) communities are able to sequence a genome themselves using 454/Illumina-Solexa/Helicos/Pacific Biosystems approaches in their own lab?  There is still a huge lag in what kinds of tools researchers can use to annotate genomes to fix gene models and add functions.  Hopefully projects like GMOD will continue to develop useful tools for solving these needs, but there is certainly a need for better support of distributed community annotation of genomes where this little direct money for supporting curators from a single place.

Trichoderma reesei genome paper published

TrichodermaThe Trichoderma reesei genome paper was recently published in Nature Biotechnology from Diego Martinez at LANL with collaborators at JGI, LBNL, and others. This fungus was chosen for sequencing because it was found on canvas tents eating the cotton material suggesting it may be a good candidate for degrading cellulose plant material as part of cellulosic ethanol or other biofuels production.  The fungus also has starring roles in industrial processes like making stonewashed jeans due to its prodigious cellulase production.

The most surprising findings from the paper include the fact that there are so few members of some of the enzyme families even though this fungus is able to generate enzymes with so much cellulase activity. The authors found that there is not a significantly larger number of glucoside hydrolases which is a collection of carbohydrate degrading enzymes great for making simple sugars out of complex ones. In fact, several plant pathogens compared (Fusarium graminearum and Magnaporthe grisea) and the sake fermenting Aspergillus oryzae all have more members of this family than does.  T. reesei has almost the least (36) copies of a cellulose binding domain (CBM) of any of the filamentous ascomycete fungi.  They used the CAZyme database (carbohydrate active enzymes) database which has done a fantastic job building up profiles of different enzymes involved in carhohydrate degradation binding, and modifications.

Whether T. reesei is really the best cellulose degrading fungus is definitely an open question.  That it works well in the industrial culture that it has been utilized in is important, but there may be other species of fungi with improved cellulase activity and who may in fact have many more copies of cellulases.  So it will be good to add other fungi to the mix with quantitative information about degradation to try and glean what are the most important combination of enzymes and activities.

One technical note.  The comparison of copy number differences employed in the paper is a simple enough Chi-Squared, work that I’ve done with Matt Hahn and others include a gene family size comparison approach that also taked into account phylogenetic distances and assumes a birth-death process of gene family size change.  It would be great to apply the copy number differences through this or other approaches that just evaluate gene trees for these domains to see where the differences are significant and if they can be polarized to a particular branch of the tree.

So will this genome sequence lead to cheaper, better biofuel production? Certainly it provides an important toolkit to start systematically testing individual cellulase enzymes. It’s hard to say how fast this will make an impact, but the work of JBEI and a host of other research groups and biotech companies are going to be able to systematically test out the utility of these individual enzymes.

There is also evolutionary work by other groups on the evolution of these Hypocreales fungi trying to better define when biotrophic and heterotrophic transitions occurred to sample fungi with different lifestyles that might have different cellulase enyzmes that may not have been observed. Defining the relationships of these fungi and when and how many times transitions to lifestyles occurred to choose the most diverse fungi may be an important part of discovering novel enzymes.

Also see

Martinez, D., Berka, R.M., Henrissat, B., Saloheimo, M., Arvas, M., Baker, S.E., Chapman, J., Chertkov, O., Coutinho, P.M., Cullen, D., Danchin, E.G., Grigoriev, I.V., Harris, P., Jackson, M., Kubicek, C.P., Han, C.S., Ho, I., Larrondo, L.F., de Leon, A.L., Magnuson, J.K., Merino, S., Misra, M., Nelson, B., Putnam, N., Robbertse, B., Salamov, A.A., Schmoll, M., Terry, A., Thayer, N., Westerholm-Parvinen, A., Schoch, C.L., Yao, J., Barbote, R., Nelson, M.A., Detter, C., Bruce, D., Kuske, C.R., Xie, G., Richardson, P., Rokhsar, D.S., Lucas, S.M., Rubin, E.M., Dunn-Coleman, N., Ward, M., Brettin, T.S. (2008). Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina). Nature Biotechnology DOI: 10.1038/nbt1403

Podospora genome published

P.anserinaThe genome of Podospora anserina S mat+ strain was sequenced by Genoscope and CNRS and published recently in Genome Biology. The genome sequence data has been available for several years, but it is great to see a publication describing the findings.  The 10X genome assembly with ~10,000 genes provides an important dataset for comparisons among filamentous Sordariomycete fungi. The authors primarily focused on comparative genomics of Podospora to Neurospora crassa, the next closest model filamentous species.  Within the Sordariomycetes there are now a very interesting collection of closely related species which can be useful for applying synteny and phylogenomics approaches.

The analyses in the manuscript focused on these differences between Neurospora and Podospora identifying some key differences in carbon utilization contrasting the coprophillic (Podospora) and plant saprophyte (Neurospora).  There are several observations of gene family expansions in the Podospora genome which could be interpreted as additional enzyme capacity to break down carbon sources that are present in dung.

The genome of Neurospora has be shaped by the action of the genome defense mechanisms like RIP that has been on interpretation of the reduced number of large gene families and paucity of transposons. The authors report a surprising finding that in their analysis that despite sharing orthologs of genes that are involved in several genome defense, they in fact find fewer repetitive sequences in Podospora while it still fails to have good evidence of RIP.

Overall, these data suggest that P. anserina has experienced a fairly complex history of transposition and duplications, although it has not accumulated as many repeats as N. crassaP. anserina possesses all the orthologues of N. crassa factors necessary for gene silencing, including RIP, meiotic MSUD and also vegetative quelling, a post transcriptional gene silencing mechanism akin to RNA interference

I think this data and observations interleaves nicely with the work our group is exploring on evolution of genome of several Neurospora species which have different mating systems. The fact that the gene components that play a role in MSUD and a RIP are found in Podpospora but yet the degree of RIP and the lack of any observed meiotic silencing suggests some interesting occurrences on the Neurospora branch to be explored.  The potentially different degrees of RIP efficiency and types of mating systems (heterothallic and pseudohomothallic) among the Neurospora spp may also provide a link to understanding how RIP evolved and its role on N. crassa evolution.

Senescence in Podospora

Another aspect of Podopsora biology that isn’t touched on, is the use of the fungus as a model for senescence.  The fungus exhibits maternal senescence which involves targeted changes in the mitochondria that leads to cell death.  The evolutionary and molecular basis for this process has been of interest to many research groups and the genome sequence can provide an additional toolkit for identifying the factors involved in the apoptosis process in this filamentous fungi. Whether it will help find a real link for aging research in other eukaryotes remains to be seen, but it is a good model system for some aspects of how aging and damage to mtDNA are linked.

Espagne, E., Lespinet, O., Malagnac, F., Da Silva, C., Jaillon, O., Porcel, B.M., Couloux, A., Aury, J., et al (2008). The genome sequence of the model ascomycete fungus Podospora anserina. Genome Biology, 9(5), R77. DOI: 10.1186/gb-2008-9-5-r77

Deep EST sequencing = RNA-Seq

The transcriptional landscape of yeast has been (further) defined with Solexa sequencing in a method deemed “RNA-Seq”, but what I would call “deep EST sequencing”.  This approach for transcriptional profiling by sequencing alone is sure to be used by many labs looking for lower and more complete ways to describe and quantitate the full population of transcripts in an organism.  

Nagalakshmi, U., Wang, Z., Waern, K., Shou, C., Raha, D., Gerstein, M., Snyder, M. (2008). The Transcriptional Landscape of the Yeast Genome Defined by RNA Sequencing. Science DOI: 10.1126/science.1158441
 

 

RIPing in an asexual fungus

ResearchBlogging.orgA.niger conidiophoreA paper in Current Genetics describes the discovery of Repeat Induced Polymorphism (RIP) in two Euriotiales fungi.  RIP has been extensively studied in Neurospora crassa and has been identified in other Sordariomycete fungi Magnaporthe, Fusiarium. This is not the first Aspergillus species to have RIP described as it was demonstrated in the biotech workhorse Aspergillus oryzae.  However, I think this study is the first to describe RIP in a putatively asexual fungus.  The evidence for RIP is only found in transposon sequences in the Aspergillus and Penicillium.  A really interesting aspect of this discovery is RIP is thought to only occur during sexual stage, but a sexual state has never been observed for these fungi.   Continue reading RIPing in an asexual fungus

(re)Annotating GenBank

NCBI LogoTom Bruns, Martin Bidartondo and 250 others sent a letter to Science describing the current problems with fixing annotation in GenBank. There is an entertaining accompanying news article that interviews several people about the problem of updating annotation and species assigned to sequences in the database. In particular the problem for mycologists that many fungi found from metagenomic approaches are only identified through molecular sequences and having the wrong species associated with a sequence can be difficult when studying community ecology composition.  This problem is not limited to fungi by any means, but recent reports find as many as 20% of fungal Intergenic Spacer (ITS) sequences are mis-attributed to the wrong species. 

There’s a nice quote in the news article from Steven Salzberg talking about the difficulties in getting sequences, especially from big centers, updated. I’m sure he is thinking of many examples, like reclassifying some Drosophila sequence traces.

Continue reading (re)Annotating GenBank

Aspergillus comparative transcriptional profiling

ResearchBlogging.org

Researchers from Technical University of Denmark published some interesting results from comparing expression across the very distinct Aspergillus species.

Kudos also goes to making it Open Access. I am posting a few key figures below the fold because I can! They grew the fungi in bioreactors fermenting glucose or xylose. After calibrating the growth curves they were able to sample the appropriate time points for comparison of gene expression across these three species. They found a set of genes commonly expressed.

Continue reading Aspergillus comparative transcriptional profiling

B. dendrobatidis strain JAM81 released

B.dendrobatidis zoosporeThe following is an announcement to the B.dendrobatidis and fungal community at large from Alan Kuo at JGI. This is the JAM81 strain (Jess Morgan collected from a frog in the California Sierra Nevada). The JEL423 (Joyce Longcore, collected in Panama) strain genome sequence and annotation is available from the Broad Institute.

Please do contact me if you would like to contribute to assigning functions to the annotation. We’re in the last round of analyses for some of the genome work, but if there are particular questions you want to contribute to, we’re open to collaborators and can outline the basis of our work to see how other work can complement it.

From Alan Kuo at JGI:

The JGI Batrachochytrium annotation portal is now on the public JGI website. As it is public, no password is required.

For those of you who have not yet registered to be an annotator, go to this new link to register.As before, please choose a username that is personal, so that other annotators may be able to recognize it as yours. A derivative of your personal name would be best.

Those of you who are already registered, you do not need to do anything. Your old pre-release username and password are valid on the new public portal too.

As always, please direct all questions and problems to me. Use email or phone: Cheers, Alan.

Some information about the assembly and annotation:

The first annotation of the 127 scaffolds and 24 Mbp of JGI’s 8.74X assembly of the Batrachochytrim dendrobatidis JAM81 genome. We predict 8732 genes, with the following average properties:

Gene length 1825.16 nt
Transcript length 1407.29 nt
Protein length 450.56 aa
Exon frequency 4.29 exons/gene
Exon length 328.37 nt
Intron length 129.18 nt
Gene density 359.1 genes/Mbp scaffold

The genes were found by the following methods:
Total models 8732 (100%)
Jason’s models 3214 (37%)
cDNAs and ESTs 518 (6%)
Similarity to nr 1928 (22%)
ab initio 3072 (35%)

The genes were validated by the following evidence:
start+stop codons 7990 (92%)
EST support 2488 (28%)
nr hit 6787 (78%)
Pfam hit 4329 (50%)