Progress 09/01/08 to 08/31/13
Outputs
Target Audience: Our target audiences include researchers who study oomycete pathogens of animals and plants, researchers who study diseases of fish, crustaceans and other animals (especially salmon and crayfish), and the aquaculture industry (especially salmon and crayfish producers). Another target audience was high school students who participated in our summer programs. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? In the area of training and development, the project sponsored a one week hands-on annotation jamboree. The 26 participants included 7 graduate students, 4 undergraduates and 2 faculty from undergraduate institutions. These participants developed greatly strengthened bioinformatics skills and greatly strengthened professional networks. A much larger number of students, particularly in collaborator Van West’s lab, participated in the genome annotation. Postdoctoral fellow Irene de Bruijn, who was co-first-author on the genome publication, made major contributions to the genome annotation and to the experimental work that supported many of the annotations. The project also sponsored two one-week summer programs for high school students centered around genomics, bioinformatics, biotechnology and aquaculture, that was attended by a total of 25 students. How have the results been disseminated to communities of interest? The sequencing of the Saprolegnia parasitica genome was published in a top, open access journal, PLoS Genetics, and several addition manuscripts are in preparation. All of the sequence resources were made publicly available at an early date at the broad Institute web site, and completed genomes have been deposited in GenBank and at FungiDB.org. Brett Tyler, Rays Jiang, Irene de Bruijn and Pieter van West have presented numerous talks and posters on the project outcomes at conferences and seminars. Pieter van West has given a number of presentations to the aquaculture industry on the findings. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
IMPACT Aquaculture production is the fastest growing sector of the world food economy. In the US salmon and catfish are major components of the aquaculture industry. Industrialization of fish and crustacean production has brought with it disease management challenges, both on farms and in surrounding aquatic ecosystems. This project generated genome sequence information for five important oomycete aquaculture pathogens, namely the salmon pathogens Saprolegnia parasitica and Saprolegnia diclina, and the crayfish pathogens Aphanomyces invadans and Aphanomyces astaci. The information revealed that these pathogens are unexpectedly vulnerable to two classes of fungicides that may prove useful in controlling these diseases. ACCOMPLISHMENTS Objective 1. Generate a whole genome shotgun assembly of salmon-pathogenic Saprolegnia parasitica isolate CBS223.65. 1) Major activities completed / experiments conducted: The strategy of whole genome shotgun sequencing was applied to S. parasitica strain CBS223.65, which is a strain isolated from infected pike. 2) Data collected: A combination of 454 and Sanger sequencing data (>50-fold coverage) were used to assemble the genome. 3) Summary statistics and discussion of results: Based on the distribution of coverage and polymorphisms, it was concluded that the assembly represents a composition of regions of diploid consensus (76%) with the remainder corresponding to separately assembled haplotypes, resulting from the high polymorphism rate. Based on read coverage it was estimated that the total genome size was 62.8 Mb. Approximately 1/3 of the assembled genome exhibits loss of heterozygosity, indicating an efficient mechanism for revealing new variation. 4) Key outcomes or other accomplishments realized. See objective 2. Objective 2. Annotate the genome with respect to genes and other features, including the generation of 10,000 paired-end EST sequences. 1) Major activities completed / experiments conducted; We performed Illumina RNA-Seq analysis of 4 developmental stages (mycelium, sporulating mycelium, cysts, and germinating cysts), as well as a time course analysis (0, 8 and 24 hours) of trout cells challenged with cysts of S. parasitica. RNA-Seq reads were mapped to the S. parasitica genome and gene annotations, and transcript abundance values were quantified. 2) Data collected: A total of 20,113 coding gene models were computationally predicted, with 3,048 pairs of coding genes assigned as alleles within separately assembled haplotype regions (Table S3), yielding an adjusted coding gene count of 17,065. 3) Summary statistics and discussion of results: Comparison of S. parasitica with plant pathogenic oomycetes suggests that the evolution of distinct patterns of gene expansion and loss in the genomes of plant and animal pathogens. The large gene families that are hallmarks of plant pathogenic oomycetes such as Phytophthora appear to be lacking in S. parasitica. Moreover, S. parasitica encodes several genes typical of animals or animal-pathogens and lacking from other oomycetes, including disintegrins and galactose-binding lectins. 4) Key outcomes or other accomplishments realized: Key outcomes were the unexpected identification of genes in the genome indicating that Saprolegnia parasitica may sensitive to azole fungicides and to fungicides that inhibit chitin synthesis. This discovery suggests that these fungicides could be used to control the disease. Objective 3. Identify conserved and rapidly diversifying genes by generating Illumina data from a highly-salmon-pathogenic isolate of Saprolegnia parasitica (N12) and an isolate of Saprolegnia diclina (It20). 1) Major activities completed / experiments conducted; The genome of S. parasitica isolate N12 (officially VI-02736) was assembled in order to identify sequence variation compared with CBS223.65. To further expand the genome sequence resources available to the aquaculture pathogen community, and to further define the gene sets employed by animal pathogenic oomycetes, the genome sequences of two additional serious oomycete pathogens were developed and released, namely of Aphanomyces invadans (strain 9901) and Aphanomyces astaci (strain AP03). 2) Data collected: Illumina (GAII) genome sequence reads and RNA sequence reads were collected from all four isolates. 3) Summary statistics and discussion of results: The S. diclina V20 genome had the following statistics: 62.89 Mb assembly size and 17,359 genes. The Aphanomyces invadans 9901 genome had a 71.5Mb assembly size and 15,248 genes. The Aphanomyces astaci APO3 genome had a 75.8Mb assembly size and 19,119 genes. The VI-02736 reads allowed us to identify 1,467,567 SNPs between the two strains, giving an average SNP rate of 3.3%. We identified four groups of genes, that had significantly elevated sequence divergence rates as compared to core ortholog genes. 4) Key outcomes or other accomplishments realized. The sequence comparisons identified hemolysin-like proteins as key virulence determinants, raising the possibility of targeting those proteins of new fungicide development. Objective 4. Promptly release all reads, assemblies, annotation, discovered polymorphisms and EST alignments to the community. Integrate the annotated genome sequence into the centralized oomycete genomics resource at Virginia Tech. 1) Major activities completed / experiments conducted: The genome sequences of all strains were released at the Broad Institute’s web site shortly after the sequences passed quality control. The Saprolegnia parasitica CBS223.65 genome and RNA sequences have been deposited in GenBank and into the current central repository for oomycete sequences, FungiDB. 2) Data collected: N/A 3) Summary statistics and discussion of results: N/A 4) Key outcomes or other accomplishments realized: The release of these sequences has enabled detailed genome sequence analysis and comparisons by the worldwide oomycete research community. In particular, Aphanomyces researchers have taken advantage of the A. astaci and A. invadans sequences to accelerate their research. Objective 5. Train community members in the use of the sequence and add value to the sequence via a combined training workshop and annotation jamboree. 1) Major activities completed / experiments conducted: A genome sequence annotation jamboree was conduced during November 15-19, 2010. This was preceded by an oomycete bioinformatics training workshop held November 11-12, 2010. 2) Data collected: N/A 3) Summary statistics and discussion of results: During the jamboree, 26 members of the oomycete research community from around the world, including 4 undergraduates, analyzed the S. parasitica reference sequence. 4) Key outcomes or other accomplishments realized: Seven graduate students, four undergraduates and two faculty from undergraduate institutions who participated in the annotation jamboree developed greatly strengthened bioinformatics skills and greatly strengthened professional networks. Objective 6. Introduce high school students to concepts in genomics and bioinformatics, and to the importance of aquaculture, via a week-long summer program each year of the project. 1) Major activities completed / experiments conducted: A one week summer program for high school students was held July 13-17, 2009 and July 12-16, 2010. Each program consisted of nine hands-on laboratory activities, lectures on scientific topics including aquaculture, and lectures on professionalism and careers. Students were expected to maintain a detailed lab notebook, and to prepare and present a 5 minute verbal summary of what they had learned. 2) Data collected: N/A 3) Summary statistics and discussion of results: 13 students attended the 2009 program and 12 attended in 2010. 4) Key outcomes or other accomplishments realized: Students obtained a greatly expanded understanding of the research enterprise, as well as knowledge of genomics, bioinformatics and aquaculture.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Jiang, R.H.Y., de Bruijn, I., Haas, B.J., Belmonte, R., L�bach, L., Christie, J., van den Ackerveken, G., Bottin, A., Dumas, B., Fan, L., Gaulin, E., Govers, F., Grenville-Briggs, L.J., Horner, N.R., Levin, J.Z., Mammella, M., Meijer, H.J.G., Morris, P., Nusbaum, C., Oome, S., Rooyen, D.v., Saraiva, M., Secombes, C.J., Seidl, M.F., Snel, B., Stassen, J., Sykes, S., Tripathy, S., van den Berg, H., Vega-Arreguin, J.C., Wawra, S., Young, S., Zeng, Q., Dieguez-Uribeondo, J., Russ, C., Tyler, B.M. and van West, P. (2013) Distinctive expansion of potential virulence genes in the genome of the oomycete fish pathogen Saprolegnia parasitica. PLoS Genetics, 9, e1003272.
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Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: The objective of this project is to develop the genome resources of for oomycete species that are pathogens of fish, namely SAPROLEGNIA PARASITICA and SAPROLEGNIA DICLINA. During the two previous project periods, a draft assembly of the S. PARASITICA genome was produced and a provisional annotation was carried out. Illumina survey sequencing of S. PARASITICA strains CBS 223.65 (the reference strain) and N12 (a salmon pathogen) were carried out to improve the quality of the reference sequence and to identify single nucleotide polymorphisms (SNPs) in each strain. In addition Illumina survey sequencing of S. DICLINA strain VS20 was carried out to characterize species-to-species variation. A genome sequence annotation jamboree was conduced during November 15-19, 2010. During this jamboree, 26 members of the oomycete research community from around the world, including 4 undergraduates, analyzed the S. PARASITICA reference sequence. The analysis prior to, during, and subsequent to the jamboree identified a number of gene families that were potentially involved in fish infection. The analyses also however identified a number of problems with the draft assembly and annotation. The most important problem was that many genes had been assembled in two copies due differences in the two alleles of the diploid organism. To resolve this problem, the Illumina SNP data was used to carefully identify the copy number of each gene in the assembly; genes with one instead of two copies were identified as incorrectly assembled. Then the assembly was repeated user new assembly software developed by the Broad Institute, namely ALLPATHS. A second problem that emerged was that S. DICLINA was founds to be much more different to S. PARASITICA than anticipated; the large differences made it impossible to accurately match S. PARASITICA and S. DICLINA genes to identify SNPs. As a result, it was decided to gather additional sequence data from S. DICLINA and to create a stand-alone assembly. Finally, many S. PARASITICA genes were found that had no homologues in any other known species; this made functional analysis of the genes very difficult. To address this problem, RNA sequence data was collected from seven additional oomycete aquaculture pathogens. PARTICIPANTS: Principal investigator: Brett Tyler provided overall project coordination, including email communication and phone conferences with the consortium participants. Co-principal investigator: Carsten Russ (Broad Institute). Supervised genome sequencing activities by the Broad Institute genome sequencing team. Sean Sykes (Broad Institute) and Sarah Young (Broad Institute) carried out the sequence assembly. Rays Jiang (Broad Institute) and Brian Haas (Broad Institute) carried out the gene prediction and functional annotation of the genome sequence. International collaborator: Pieter van West (University of Aberdeen, United Kingdom). His laboratory characterized the SAPROLEGNIA PARASITICA strain to be sequenced and prepared DNA from S. PARASITICA for genome sequencing, using protocols provided by the Broad Institute. International collaborator: Javier Dieguez-Uribeondo (Real Jardin Botanico CSIC, Spain). Provided advice on selection of oomycete aquaculture pathogens for sequencing. Partner Institutions included: The Eli and Edythe L. Broad Institute of Harvard and MIT (Cambridge Massachussetts) University of Aberdeen (United Kingdom) Real Jardin Botanico CSIC, Spain In the area of training and development, the project sponsored a one week hands-on annotation jamboree. The 26 participants included 7 graduate students, 4 undergraduates and 2 faculty from undergraduate institutions. TARGET AUDIENCES: Brett Tyler presented one talk at an international conference on the project outcomes: Plant and Animal Genome conference January 2011. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Increased knowledge of the biology of oomycete aquaculture pathogens resulted from the analysis of the genome sequence. In particular, genes were identified that were similar and different than those used by oomycete plant pathogens. Genes were identified that could potentially be targeted by azole fungicides. SAPROLEGNIA species were found to be much more genetically diverse than previously realized. Seven graduate students, four undergraduates and two faculty from undergraduate institutions who participated in the annotation jamboree developed greatly strengthened bioinformatics skills and greatly strengthened professional networks.
Publications
- No publications reported this period
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: The objective of this project is to sequence the genomes of two oomycete species that are pathogens of fish, namely SAPROLEGNIA PARASITICA and SAPROLEGNIA DICLINA. During the previous project period, the following sequence data were generated: (i) 20fold coverage with 454-Titianium fragment reads (450 bp length); (ii) 5fold coverage with 3.5kb mate pair (jumping) reads. During this project period, additional sequence data was generated, namely 0.3fold coverage with 40kb mate pair reads (fosmids). Using these data, a provisional assembly was created using the Newbler and Arachne assemblers. The assembly consisted of 4123 contigs comprising 48.1 Mb. The contigs were assembled into 1442 scaffolds comprising 53.1 Mb. In order to facilitate gene calling and functional annotation, RNA sequence data was obtained by Illumina sequencing of cDNA from RNA extracted from different life stages of S. PARASITICA (i.e. cysts, germinated cysts, mycelium and sporulating mycelium), including various infected host tissues (i.e. whole fish, fish eggs and fish cell line). Approximately 18,000 genes were identified in the genome sequence of S. PARASITICA, and assigned predicted functions. The project sponsored a one week hands-on genomics and bioinformatics camp that was attended by 12 high school students. The camp included activities on aquaculture as well as on genomics and bioinformatics. PARTICIPANTS: Principal investigator: Brett Tyler (Virginia Tech) provided overall project coordination, including email communication and phone conferences with the consortium participants. Sucheta Tripathy (Virginia Tech) set up a bioinformatics web site to facilitate analysis of the S. PARASITCA genome sequence. Outreach coordinator Kristy Collins (Virginia Tech) supervised the organization of the high school genomics and bioinformatics camp. Outreach assistant Kris Monger organized the high school genomics and bioinformatics camp. Co-principal investigator: Carsten Russ (Broad Institute). Supervised genome sequencing activities by the Broad Institute genome sequencing team. Sean Sykes (Broad Institute) and Sarah Young (Broad Institute) carried out the sequence assembly. Rays Jiang (Broad Institute) and Brian Haas (Broad Institute) carried out the gene prediction and functional annotation of the genome sequence. International collaborator: Pieter van West (University of Aberdeen, United Kingdom). His laboratory characterized the SAPROLEGNIA PARASITICA strain to be sequenced and prepared DNA from S. PARASITICA for genome sequencing, using protocols provided by the Broad Institute. International collaborator: Javier Dieguez-Uribeondo (Real Jardin Botanico CSIC, Spain). Provided advice on selection of strains of S. PARASITICA for sequencing. Partner Institutions included: The Eli and Edythe L. Broad Institute of Harvard and MIT (Cambridge Massachussetts) University of Aberdeen (United Kingdom) Real Jardin Botanico CSIC, Spain In the area of training and development, the project sponsored a one week hands-on genomics and bioinformatics camp, similar to the previous year, that was attended by 20 high school students. The project included activities on aquaculture as well as on genomics and bioinformatics. TARGET AUDIENCES: Project staff presented a poster at the Plant and Animal Genome Conference in January 2010 and a talk at the Oomycete Molecular Genetics Meeting in Toulouse, France in June 2010. High school students were targeted via a one week, residential camp featuring lectures and hands-on activities in aquaculture, genomics and bioinformatics. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The current project is still in an early phase and no outcomes or impacts are expected from the genome sequence at this stage. The 12 high school students who attended the genomics and bioinformatics camp developed a greatly increased appreciation of how scientific research is conducted, and of how genomics and bioinformatics can contribute to agriculture, aquaculture and medicine.
Publications
- No publications reported this period
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