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IRGSP Working Group Meeting, December 19, 2002
Mita Kaigisho
Tokyo, Japan
This meeting took place the day after the very successful Commemoration
of the Completion of the High Quality Draft Sequence of the Rice Genome (http://rgp.dna.affrc.go.jp/IRGSP/Dec18_NEWS.html).
Executive Summary
Sequence-based physical maps of each chromosome based on the phase
2 sequence have been prepared by the RGP and distributed to each group. These maps are to be returned to the RGP with corrections
and appropriate annotations by January 8, 2003. Each
group is responsible for preparing a contiguous sequence (pseudomolecule)
for their assigned region(s), and the maps will be held at the RGP. A committee with representatives from TIGR, the RGP, and
Genoscope will formulate standards for construction of pseudomolecules.
There is the possibility that U.S. groups may obtain money for
finished sequencing beyond their currently assigned chromosomes. The IRGSP agreed that chromosomal assignments would be
reassessed based upon need and available resources to accommodate the additional
capacity.
About 2150 BACs/PACs that are not at phase 3 remain to be finished. The end of 2004 was agreed upon as a tentative date for
the completion of the finished rice sequence.
A new listserver will be started so that finishers can compare
notes and ask for advice with difficult regions.
Francis Quetier chaired a productive session on plans for a joint
paper on the phase 2 sequence. The paper will focus
on the map-based sequence and synteny with other cereals.
The next meeting will be February 5, 2003 in Tsukuba to be held
in conjunction with the Rice Genome Workshop and the Rice Genome Forum XI. A following meeting is tentatively scheduled for mid-May
in Shanghai.
Status of the sequence-based physical map.
Jianzhong Wu reviewed the efforts of the RGP to close gaps. The primary technique is to use PCR primers to screen
BAC and PAC libraries. PAC library, Sau3AI 71,040 clones, BAC library, MboI
55,426 clones, no end sequences are available for either library. They have achieved 96% coverage for their six chromosomes.
The RGP attempted to make psuedomolecules for each of the 12 chromosomes
based on sequence deposited in databanks. This information
- which graphically shows the RGP YAC physical map and the CUGI BAC physical
map, mapped markers, and sequenced BACs and PACs, the extent of sequence
overlap, and possibly mis-assigned clones - was distributed to all members
on December 9 for comment.
For chromosomes 3 and 10, Dick McCombie reported that resolution
of conflicts has already been sent to the RGP. Dick
says that the US groups will annotate evidence for sequence overlaps and
marker information and send that as well.
Bin Han presentated the physical map of chromosome 4. He said that his group has been
able to reduce the number of gaps from seven to three. Mr.Huang
Yuchen from Bin’s lab is currently at the RGP and screening for clones to
fill the remaining gaps. Bin said that he believed
that available clone resources-one PAC library, three BAC libraries and two
10kb insert libraries were sufficient for these purposes.
Teh-Yuan Chow reported that ten gaps remain on chromosome 5. He brought a CD with a corrected and annotated CD that
he gave to the RGP.
Akhilesh reported that for his portion of chromosome 11 there remained
seven large gaps of which five appear to be covered by the YAC map.
On another part of chromosome 11 claimed by the PGIR, Jo Messing
reported that they closed the single gap with sequence from a Monsanto clone which was missing its trace files.
For chromosome 12, Francis Quetier reported that four gaps remain
for which they have exhausted their available clone resources.
The group agreed on a Deadline of January 8 to return comments
to the RGP, with the level of detail that the RGP had employed, on the actual status of the physical map for the regions for
which they have responsibility.
Finishing Issues:
The U.S. Department of Agriculture and the U.S. National Science
Foundation have jointly requested proposals by U.S. scientists for finished
sequencing of the rice genome. This potential source
of funding would help ensure completion of the finished rice sequence and
might speed its completion. It is a resource that
the IRGSP should not ignore. The problem is that,
according to our recent surveys, all members report that they have received
commitments from their funding agencies to support finished sequencing for
the chromosomal regions that they have claimed. Dick
McCombie, speaking for U.S. sequencers, said that if they received support
for additional sequencing capacity that they could sequence BACs from any
region and only desired to speed the process. The
IRGSP agreed that, depending upon needs, chromosomal claims would be adjusted
to according to the level of additional support.
It is estimated that there remain about 2150 BACs that are not
in phase 3. We asked several of the labs what their
rate of finishing was. Francis Quetier reported that
they planned on finishing their remaining BACs by the end of 2003. The RGP reported a current rate of finishing of 30 BACs
a month, however finishing activities competed with phase 2 sequencing. They now expect that rate to double given the current
level of support. Dick McCombie estimated that CCW
could finish 10 BACs per month and that TIGR could finish about 30 per month. The IRGSP tentatively agreed that the end of 2004 might
be a reasonable tentative deadline for the completion of finished sequencing. This date will be revisited once there is more data on
the rates of finishing.
We then discussed plans and proposals for gap filling.
Francis Quetier made the following proposal for gap filling using
the Syngenta sequences:
1) BLAST Syngenta contigs against the IRGSP draft to identify the
Syngenta contigs without matches.
2) Assign chromosomal locations for these unmatched contigs using
pachytene hybridization and probing BACs and PACs. Francis
cautioned that care had to be taken in choosing probes not to use repeated
sequences and organellar sequences. Rob Martienssen
also warned about false assemblies contributing to the unmatched contigs.
3) Francis proposed a division of work where one group performed
the BLASTs. Genoscope will proceed with this task. He also suggested that other groups choose probes, perform
the pachytene hybridizations, and the filter hybridizations.
Takuji Sasaki spoke about the RGP resources for identifying gap
filling clones. There are three options: distributing
libraries, inviting researchers to the RGP to perform their own screening,
or sending flanking sequences to the RGP who can prepare primers and perform
limited screening for other groups. Takuji said that
his group much preferred the latter two options due to the difficulties and
expenses of clone distributions and invited other groups to take advantage
of the latter two options.
Rod Wing reports that ACWW has no difficulties in providing access
to libraries, clones and filters for all the libraries mentioned for gap
filling.
Dick McCombie mentioned the importance of using multiple libraries
to complete unfinished sequences. Bin Han said that
he believed that available clone resources - one PAC library, three BAC libraries
and two 10 kb insert libraries - were sufficient for these purposes.
There was wide agreement that all of the remaining gaps need to
be sized. However, Rod Wing suggests that more effort
be expended in selecting gap filling clones. Therefore
the gaps that need to be measured might be prioritized so that the most recalcitrant
gaps are measured first. Jiming Jiang has been contacted and is willing to
help but available personnel and funding are limiting. Another
possible resource is his former student, Zukuan CHEN (Institute of Genetics
and Developmental Biology, CAS, Beijing) who is receiving some support from
Bin Han for this purpose. Rod Wing suggests that Scott
Jackson, a former student of Jiming’s and an assistant professor at Purdue,
is another possibility. The number of gaps that need
to be measured is not known for the moment but Jianzhong Wu hopes to provide
an estimate after all groups have returned their physical map information.
Dick McCombie also mentioned how helpful it was for the finishers
in the CCW to compare their problems with each other in weekly group phone
calls. It was agreed to start a new listserver
for finishers so that they could post their specific problems and answers.
Next meetings:
The sequencing workshop will be held in Tsukuba on February 5. We hope to have a half day public meeting
in the afternoon and be able to meet in a smaller group using the round table
format in the morning and possibly the evening of the same day, but this
plan needs to be approved by the STAFF Institute. It
was agreed that the format of the public meeting would be reports analyzing
different aspects of the whole genome rather than individual group reports.
Jianzhong Wu agreed to be the first speaker and report on the physical map. Bal Antonio told us that he needs to have a schedule of
speakers two weeks prior to the meeting. Information
on the Rice Genome Workshops and Forum XI can be found at http://rgp.dna.affrc.go.jp/rgp/rgmXI/
Bin Han invited the IRGSP to have their next meeting in Shanghai
in May in conjunction with a meeting he is hosting on rice functional genomics. That meeting is scheduled for May 12 to 14 and the IRGSP
meeting might be held on May 15, but this date has not been finalized.
Action Items:
Graphical representations of rice chromosomal sequences need to
be returned to the RGP by January 8, 2003.
New IRGSP listserver for finishers.
A committee composed of representatives from the RGP, TIGR and Gensocope will be responsible for formulating the guidelines for the construction of the pseudomolecules.
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