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Report of the Working Group Meeting
7 February, 1999
Tsukuba, Japan
New Members
Six new members were welcomed to the Working Group:
Yue-ie Hsing reported that Taiwan has funding starting from this month to begin sequencing on chromosome 5., starting from the short arm. They plan to make a physical map using a combination of PACs and BACs. Their preliminary targets are storage protein genes, xa-5 and xa-13, and lea1.
Guo-Liang Wang reported that Singapore, upon the final approval of the Singapore government, will have two ABI 377s and one postdoc and 2 technicians to begin work on chromosome 11, the site of numerous resistance genes.
Apichart Vanavichit reported that Thailand will have money from the Royal family to begin work on chromosome 9. They have previously prepared a contig of 450 kb with BACs from another strain of japonica surrounding a flooding tolerance locus, but will sequence Nipponbare.
Villoo Morawala-Patel from Banglore, India expects to begin rice sequencing with a combination of public and private money until the Indian government works out the modalities of its commitment to the IRGSP. Her group will join one of the other members of the Working Group in sequencing a chromosome already underway. Eventually, the group would like to begin sequencing chromosome 8.
Michel Delseny reported that France will sequence chromosome 12. Combined laboratories from ORSTAM (IRD), CIRAD, and CNRS at the University of Perpignan have begun work on the Project by constructing a 400 kb BAC contig. They will be joined later this year by Genoscope, where the bulk of the sequencing will be carried out.
Tom Bureau ( Montreal, Canada) reports that so far his laboratory has received equipment money to set up a rice genome sequencing laboratory at McGill University. He expects to coordinate his efforts with some other national group on a chromosome already underway.
Progress
The Rice Genome Research Program (RGP) is preparing PAC-based physical maps of chromosomes 1, and 6 for their own sequencing effort and chromosome 5 for Taiwan. They are using mapped sequences to anchor these contigs. Takuji Sasaki reported that the RGP had screened half of their PAC library with all available mapped probes on chromosome 6, 81 STSs generated by RFLP markers and 152 ESTs, and half of the available markers (61 STSs generated by RFLP markers and 113 ESTs) on chromosome 1. So far, the results point to an uneven distribution of genes: On chromosome 6, 29 Mb is covered by YAC contigs, but only 12 Mb is covered by anchored PAC contigs. High volume genomic sequencing has also begun at the RGP. Sasaki pointed out difficulties of G and AT repeats that contributed to an inability to maintain an average Phred score of 70 across a PAC. The RGP will send clones of the PAC library to the Clemson Genomics Institute (CUGI) for distribution to sequencing centers in the US and to Mike Bevan for use by the EU rice sequencing effort.
Guofan Hong (Shanghai, China) reported that 50 BAC contigs provide 90% coverage on chromosome 4. They have started sequencing on six of these. They have obtained about 700 kb of sequence from 5-6 BACs in different contigs. Annotation problems delay the release of this sequence.
Jo Messing (Rutgers, US) reported progress of the coordinated efforts between his lab and Rod Wing's lab to use ESTs to map BACs to chromosome 10. Jo also mentioned the recently announced United States Rice Genome Sequencing Project, and this was discussed further by Ed Kaleikau (USDA).
Rod Wing (Clemson, US) reports that CUGI will send copies of their HindIII BAC library to all sequencing centers upon request. BAC libraries have already been provided to France (P. Lagoda, CIRAD), UK (I. Bancroft), Japan (N. Kurata), Korea (M.Y. Eun), and in the US, Rutgers (J. Messing) and U. Georgia (A. Paterson). Additional requests have been received from Japan (T. Sasaki), Singapore (G.L. Wang), and Thailand (A. Vanavichit). Rod requested that the addresses of BAC clones identified by hybridization with chromosome specific probes be returned to CUGI so that this information can be entered into their database.
Dick McCombie (Cold Spring Harbor, US) talked about sequencing presumed overlapping BACs from indica and japonica cultivars. One of the key features of these clones is the number of repeated elements encountered and the difficulty they posed for DNA sequencing.
Mike Bevan (John Innes, EU) talked about exploratory sequencing done by EU groups of a 500 kb contig on chromosome 2 comprised of BACs from various libraries. To date, 29 genes have been found in 130 kb of sequence analyzed. Among the difficult features encountered were hairpin loops. They are using ESTs to map Nipponbare BACs to chromosome 2 with anticipation of EU funding as early as October.
Moo Young Eun (Suwon, Korea) reported preliminary work on selection of Nipponbare BACs from two regions of chromosome 1. Three to four BACs from each region are being subcloned using restriction enzymes and sonication. One Mb of sequencing of these BAC contigs will be carried out each year.
Database
After Progress reports from each of the active members of the Working Group, Katsumi Sakata demonstrated INE, a prototype of the proposed Rice Sequencing Database. A preliminary version will soon be available from the RGP home page. The database was developed with the Java programing language and shows graphics and text in a map oriented fashion. Currently the following objects are related to each other in the database: genetic maps, YAC and PAC physical maps, information on individual clones, and sequence data.
Jo Messing suggested that the database have the ability to indicate which group had contributed information.
Mike Bevan suggested that the names of the clones in the BAC and PAC libraries be standardized so that the names meant the same thing to everyone.
In response to a question from Dick McCombie, Sakata replied that he would be able to add a function to the INE database to answer relational queries in about three months.
The database can be of great use in coordinating the work of the individual sequencing efforts. The database group will post the clones that are being sequenced, or have been tiled in preparation for sequencing by each sequencing group. It was decided that each sequencing group will be responsible for posting a web page that contains the names of clones being mapped or sequenced. These clones or the contigs they belong to must be identified by two mapped ESTs. Sequence information, as it becomes available, will also be posted on the individual web sites. The RGP will be provide a standardized format for these pages to facilitate the compilation of data into the centralized database.
Guidelines
1) Under Accuracy, the third paragraph has been modified:
"The Rice Genome Sequencing Project will adopt the standards of The Human Genome Project, established at its Bermuda meetings in 1996 and 1997, which has agreed to accept a standard of less than one error in 10,000 bp. While the level of accuracy is difficult to verify, this standard is typically achieved by a combination of high quality shotgun sequence reads, appropriate redundancy, and the insistence that at least 97% of all bases be sequenced on both strands or on multiple templates using two chemistries. Minimum error estimation values provided by PHRAP of 40 are consistent with this level of accuracy. Less than 1% of the sequence with a PHRAP value of less than 40 is permitted, regions that fall below this value should be indicated. Further, restriction sites predicted from the sequence must conform to observed digest patterns."
2) Under Annotation, insert a second sub-heading reading, "A contig will be considered complete when no more clones can be found from available resources."
3) Under Outreach, add a sentence reading, "The IRGSP welcomes the participation in its activities of all scientists who can contribute to its goals." Also, RICE GENOME will be changed to ORYZA.
Whole Genome Sequencing
Mike Bevan suggested that the Working Group reconsider whole genome sequencing as an approach to rapidly complete the rice genome sequence. The method would sequence a 10X coverage of both ends of 2 kb shot gun clones prepared from the entire rice genome rather than from individual BACs. Both ends of a smaller number of larger insert random clones would also be sequenced. Software under development would be used to assemble these sequences. It is presumed that this would reveal the sequences for all of the unique regions in the rice genome. These small contigs would be anchored to the genetic map with BAC end sequences that are already being produced. Where practical, gaps would be completed using conventional finishing strategy. Bevan estimates that the first phase of the project would take $50M to complete.
Subsequent discussion centered on the likelihood of the assembly working, whether the gaps would be in uninteresting repeated sequence regions, and the necessity of obtaining a 'complete' sequence of the rice genome.
Three Year Projection
Each of the national groups was asked to project their finished sequence production for three years.
| Japan | 60 Mb |
| EU | 25 |
| US | 24 |
| France | 30 |
| China | 15 |
| Singapore | 3 |
| Korea | 3 |
| Taiwan | 4 |
| Thailand | 3 |
| Canada | 3 |
| India | 3 |
| Total | 173 Mb |
Attendees:
Dr. Takuji Sasaki, RGP / NIAR, Japan
Dr. Ben Burr, Brookhaven Nat. Laboratory, U.S.A.
Dr. Michael Bevan, John Innes Centre, U.K.
Dr. Rod Wing Clemson, Univ. Genomics Inst., U.S.A.
Dr. Richard McCombie, Cold Spring Harbor Laboratory, U.S.A.
Dr. Joachim Messing, Rutgers Univ., U.S.A.
Dr. Gou Fan Hong, Nat. Center Gene Research, China
Dr. Michel Delseney, Perpignan Univ., France
Dr. Moo Young Eun, Nat.Inst. Agric. Science & Tech., Korea
Dr. Yue-ie Hsing, Academia Sinica, Taiwan
Dr. Guo-liang Wang, National Univ. Singapore
Dr. Thomas Bureau, McGill Univ., Canada
Dr. Apichart Vanavichit, Nat. Center Genetic Engineering & Biotech., Thailand
Dr. Villoo Morawal-Patell, Nat. Center Biological Sciences, India
Observers:
Dr. Pieter de Jong, Roswell Park Cancer Institute, U.S.A.
Dr. Ed Kaleikau, USDA / NRI
Dr. Andrew Paterson, Georgia Univ., U.S.A.
Dr. Pat Heslop-Harrison, John Innes Centre, U.K.
Dr. Patrick Schnable, Iowa State Univ., U.S.A.
Dr. Frances Burr, Brookhaven Nat. Laboratory, U.S.A.
Dr. Choong Hyo Yun, Nat.Inst. Agric. Science & Tech., Korea
Dr. Ryu Ohsugi, MAFF, Japan
Dr. Yoshiaki Nagamura, RGP
Dr. Takashi Matsumoto, RGP
Dr. Kimiko Yamamoto, RGP
Dr. Tomoya Baba, RGP
Dr. Hiroyoshi Aoki, RGP
Dr. Katsumi Sakata, RGP
Dr. Bal Antonio, RGP
Posted March 10, 1999 by B. Burr
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