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Groups are invited to apply to participate in the sequencing of the ~ 5 Mbp genome of Xanthomonas axonopodis pv citri, the causative agent of citrus canker.
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Background |
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General organization of the sequencing effort |
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Bioinformatics |
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Sequencing Strategy |
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Chronogram |
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Sequencing Efficiency Expectations |
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Financing the Project |
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Applications |
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Deadline |
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Forms |
In Brazil and around the world, Xanthomonas axonopodis pv citri causes citrus canker in citrus plants with an annual economic impact estimated at tens of millions. Many other related Xanthomonas species attack other economically important crops such as rice, cotton, beans, and grapes. Knowledge of the complete sequence of the X. axoponopodis pv citri genome will therefore be of enormous potential value to both the Brazilian internal economy and numerous agricultural sectors in a large number of countries worldwide. The genome database generated by this project will be an important resource in efforts to understand Xanthomonas biology in general and the variety of Xanthomonas-host interactions of economic consequence. Furthermore, this genome sequence will be of tremendous use in basic research into the molecular basis of plant-pathogen interactions since the closely related X. campestris pv campestris is currently being used as a model pathogen in studies with Arabidopsis thaliana. Arabidopsis, whose genome will be completed shortly, is the model organism for studies of plant genetics. Finally, X. axoponopodis is a close relative of Xylella fastidiosa, but has a genome of approximately twice the size. (It is estimated that the genome of X. axonopodis pv citri has approximately 5 Mbp.) The availability of the two genomes will allow us to compare the genetic makeup of these two species and gain insight into the important similarities and differences which determine their unique metabolisms, life cycles and interactions with their common host.
General organization of the sequencing effort
The sequencing of the X. axonopodis pv citri genome will be carried out by a maximum of 12 labs: 2 central sequencing labs and up to 10 smaller sequencing labs. One of the central laboratories will be located in the Biochemistry Department of the Institute of Chemistry at the University of São Paulo (USP) and will be coordinated by Fernando C. Reinach, Ana C. R. da Silva, Ronaldo B. Quaggio and Shaker Chuck Farah. The second central lab will be coordinated by Jesus A. Ferro at the Technology Department of UNESP in Jaboticabal. These laboratories will divide the coordination of library generation, mapping of clones and assembly of the genome as described below. The other sequencing laboratories will be selected by a steering committee nominated by FAPESP based on their previous genomic sequencing experience. New groups will be selected based on their interest in joining the ONSA network for this high-throughput project.
Bioinformatics for this project will be administered via a two-tiered approach. At the upper tier will be the Laboratory for Bioinformatics (LBI) of Unicamp, coordinated by João C. Setubal and João Meidanis. At the lower tier, there will be two small bioinformatics labs in each of the central sequencing labs in São Paulo and Jaboticabal. These small bioinfo labs will divide the tasks of storing, processing, assembly, and annotation of DNA sequences using the software developed by the LBI for the Xylella fastidiosa sequencing project.
In addition to overall bioinformatics supervision, the LBI will provide personnel training, coordinate annotation of the genome and produce a self-contained and portable database with project results. Furthermore the LBI will be responsible for generating the software for scaffold assembly of the physical map (see below) and for comparison of the final sequence with sequences from related genomes (Xylella fastidiosa and other Xanthomonas species).
Sequencing will be performed in two stages:
Stage 1: End sequences will be obtained from one thousand independent cosmid clones and 15,000 plasmid clones (30,000 reads = 3 X coverage of the genome). This data will be assembled and the cosmid end sequences will be used to order the contigs generated by the shotgun library into a scaffold of the physical map. From this point it will be possible to identify how many gaps remain in the physical map.
Stage 2: A minimum set of cosmid clones (approximately 150-200) that cover the contigs identified in stage 1 will be distributed for sequencing. One of the central sequencing labs will be responsible for coordinating the sequencing and assembly of these cosmids. In parallel, the second central sequencing lab will be responsible for identifying and characterizing clones which span the remaining gaps in the physical map. For this purpose a combination of PFGE mapping, PCR closure and screening of BAC and lambda libraries will be used. These clones will then be distributed for sequencing, thereby completing the sequence of the genome.
December 1999: End of stage 1.
December 2000: Deadline for finishing of the sequencing of the first set of cosmids.
July 2001: Deadline for finishing the genome sequence.
Sequencing Efficiency Expectations
Our aim is to obtain a high throughput of DNA sequencing and annotation. Based on an estimated genome size of 5 mega base pairs, we expect to sequence between 150 and 200 cosmid clones with an average size of 40,000 bp each and possibly some BAC clones (approx. 100,000 bp in size). We also plan to shotgun-sequence the genome to at least 3X coverage (15,000,000 bp).
Each sequencing group will be
expected to generate an absolute minimum of 250,000 finished
bases (from cosmid or BAC) per year for two years. In addition,
in the first six months, each group will be expected to produce
3,000 shotgun reads with at least 400 bases of quality 20
(according to the phred program definition) or better.
Note: 250,000 finished bases represent ~6 finished
cosmids. A 96-lane machine running once a day can close a cosmid
every two weeks, or 24 cosmids a year. Thus, the absolute minimum
work expected is only one quarter of that. Each sequencing lab
applicant must indicate in its application form how many cosmids
the lab expects to be able to sequence per year (being 6 the
absolute minimum).
The payments for sequencing will be made according to the following general guidelines:
There are two kinds of applications:
The role, expected work and payment of sequencing labs has already been described above.
There will be 2 Bioinformatics Associate positions. These associates should be faculty members or persons with a Ph.D. with expertise in software system design and implementation. Expertise in bioinformatics is desirable but not essential. The goal is to have one of the associates work with the USP/IQ Central Sequencing Lab, and the other with the UNESP/Jaboticabal Central Sequencing Lab, both in collaboration with and under the supervision of the LBI. In the case of faculty members (i.e., persons already affiliated with an academic institution), geographic proximity with the respective sequencing lab is important. In the case of unaffiliated persons with a Ph.D., the daily working place will be the assigned lab; and a FAPESP postdoc fellowship will be provided.
Applications to participate in the Xanthomonas Genome Project as a Sequencing Laboratory or Bioinformatics Associate will be received by FAPESP up to July 8th, 1999
Selection of groups by the steering committee will be made by end of July, 1999.
Contracts will be signed by early August, 1999.
