Round Table Discussion
Tsukuba, Japan
February 5, 2003


Executive Summary

Greater than 94% of the Nipponbare genome is covered by public sequence. About 40% of this sequence is finished quality. Approximately 2000 clones, including those that fill gaps, remain to be completed with finished quality sequence.

The IRGSP established a plan for completion of the finished quality sequence for the rice genome which includes the following steps:

Finishing Status

Jianzhong Wu: Summarized the sequence-based physical maps. See the table below that gives the summary of the physical maps returned to the RGP as of January 8, 2003. These maps show 94% coverage based on the revised IRGSP estimates of chromosome size. This table and two graphical maps show 88 physical gaps not counting the telomere gaps. All of the latter are expected to be small. Based on fiber-FISH experiments, those on chromosome 1 and 4 are less than 50 Kb, the sizes of those on 10 were measured to be 40 and 80 kb.
Chromosome Chrom. Tile Tiling Path Chromosome Gaps Gap Size

Length Mb Length Mb BACs/PACs % Coverage
Mb
1 45.2 43.5 387 96.2 7 1.7
2 37.3 36.4 355 97.6 4 0.9
3 41 36.6 312 89.3 19 4.4
4 36 35.2 298 97.8 3 0.8
5 31 29.2 276 94.2 8 1.8
6 32.6 31.2 279 95.7 2 1.4
7 30.7 29.7 290 96.7 3 1.0
8 29.6 28.7 284 97.0 2 0.9
9* 23.6 22.9 216 97.0 5 0.7
10 23 22.6 201 98.3 8 0.4
11 30 24.1 218 80.3 22 5.9
12 30 27.2 270 90.7 5 2.8
Totals 390 367.3 3386 94.2 88 22.7
* An estimated 3 Mb of rDNA repeats have been subtracted from the chromosome 9 length.

It is the experience of the members that Syngenta sequence helps to extend contig ends for less than 50% of the gaps. This sequence needs to be used with caution because when the reads that underlie the Syngenta contigs have been trimmed the contigs frequently cannot be reassembled. The Syngenta sequences can be used to provide coverage for small gaps, but has not proved useful for gaps larger than 5 kb.

Takashi Matsumoto summarized the RGP progress on chromosomes 1, 2, 6, 7, 8, and 9. On these chromosomes he reported 24 clones at phase 1, 1106 clones at phase 2, and 622 phase 3 clones. They expect to have a finishing rate of 50 BACs per month and plan to concentrate on chromosomes 7 and 8 with the goal of finishing these two this year.

Takashi described refinements in their sequencing strategy which allowed them to increase the pace of finishing in problem areas. For bridge clones they changed chemistry, used sonicated templates, transposon primers, and the "TempliPhi (Amersham)" system. Where they had no clones they used upstream primers and BAC templates.

Bin Han reported that on chromosome 4, seven physical gaps had been reduced to three by Huang Yuchen working at the RGP to identify clones. The remaining three comprise less than 500 kb in total. Bin used the indica sequence he has obtained from chromosome 4 to look for gap filling clones and found that the gaps were also present in the libraries from both subspecies.

The centromere of chromosome 4 has been sequenced. Of 293 total BACs, 245 are finished and 48 BACs remain to be finished.

Bin has additional finishing capacity and an established track record for finishing. He would have to apply for additional funds to work on other chromosomes.

The question of using support that Bin Han gives to Zukuan Chen to use fiber-FISH to measure gaps on other IRGSP chromosomes. Bin said that this could be done but must be in collaboration with Jiming Jiang.

Rod Wing reported that the chromosome 10 manuscript will be submitted next week. On Chromosome 3S ACWW has the first 65 cM or about 14.4 Mb. Of this 72% of the BACs are finished, 13% in one contig, and 15% are ordered. They expect to finish all of the identified BACs by this May. Funding for finishing all chromosome 3S BACs, including those to fill remaining gaps, is in hand.

There remain eight gaps in this region and he estimates that four of these are physical gaps. Rod said that they will use unique end sequence to screen all available libraries (their two 10kb libraries and the BAC, PAC filters) to fill and sequence these gaps. He said that he is planning to make a 10X random sheared fosmid library (40kb size) to assist the IRGSP in gap filling. The library should be available in three months.

Rod urged the RGP to distribute its BAC library so that it could be available for more efficient screening by IRGSP members. He offered to distribute these clones or filters to US members of the IRGSP if the clones could be made available. Takuji Sasaki agreed to make the libraries available.

They have also been working on the telomere gaps genome-wide and have isolated candidate telomere regions from 3-4 telomeres.

They have two pending proposals for money to finish more BACs, one is to USDA for finishing 66 BACs in one year as well as possibly filling gaps. Rod is talking with Akhilesh Tyagi and they may pick up BACs from India's region on chromosome 11. The second proposal is to NSF and is for finishing 300 140Kb BACs as well as 50 difficult BACs from anywhere in the genome. The NSF proposal also includes a training component and workshop.

Rod also explained that he thought it was time to consider the finishing problem without consideration of chromosomal boundaries. He proposed that as we get nearer to the deadline of December 2004, the IRGSP will need to be flexible and have to respond rapidly to finishing in problem areas. Rod suggested that microtitre plates be arrayed and distributed with unfinished BACs so that these could be accessed as needed.

Robin Buell reported that they currently have 25 Mb of sequence for chromosome 3. There are 8 physical gaps plus the centromere and the telomere on the long arm. She reported 57 finished BACs, 115 in closure, and 11 in shotgun. She said that the first two gaps had resisted all efforts to identify gap-filling clones, but had not tried the RGP clones. On chromosome 11, the TIGR areas comprise 12.4 Mb with five gaps including the centromere. In this region there are 72 BACs in closure and 15 BACs in library/shotgun. Jiming Jiang will to the fiber-FISH work to measure the physical gaps for which no clones can be found.

There is no current funding to finish the chromosome 11 BACs or the estimated 3 Mb of gaps in TIGR's regions on 3. TIGR has applied for funds for finished sequencing from both the USDA and NSF.

Robin also is preparing a single read Syngenta library database for IRGSP members to query.

Robin also suggested doing low coverage sequencing on BACs from unmapped contigs to identify gap-filling contigs.

Francis Quetier reported that for chromosome 12 they had sequenced 27.2 Mb out of an estimated 30 Mb covered by 258 BACs. All but two of these have been sequenced to finished quality. On this chromosome there are 4 remaining gaps plus the centromere.

Francis reported that Syngenta draft sequences give various results for gap filling: no match at all, extension of both ends, or extension of one end only. Francis will contact Angelique D'Hont to ask her whether she could do BAC FISH and Fiber FISH to measure the size of remaining gaps and what would be the conditions for doing this.

Akhilesh Tyagi reported that there are 8 gaps not yet filled in India's region on chromosome 11 but that they had identified 4 new BACs to fill the other 4 gaps. He will send a person to the RGP to identify more gap-filling BACs. India's region between 57.3 cM and 116.2 cM comprises about 12 Mb and 117 identified clones. Thirty of these clones are now in finishing and he will send a person to Dick McCombie's lab learn how to finish difficult sequences.


Teh-Yuan Chow reported that on chromosome 5 Taiwan has 9 contigs that comprise 276 BACs. Of these, 20 are in phase 1, 220 are in phase 2 and 36 are finished. They have money to finish these clones and expect to do so at the rate of 20 BACs per month. Of the 8 physical gaps, 5 are very small and 3 are also gaps in the YAC-based physical map. They will send someone to the RGP to use their facilities to identify gap-filling BACs.

Jang Ho Han reported that Korea's region on chromosome 9 contained no physical gaps and contained 19 BACs which they expected to finish by the end of the year.


Antonio Costa de Oliviera reported that Brazil was still sequencing two BACs comprising 244 Mb on chromosome 9 and had been having problems preparing shotgun libraries. He proposed working on an additional 8 clones from this region.


Summary and a Plan for Finishing the Nipponbare Genome

Group BACs to
Finish 		Claimed
 Gaps
Questionable
Funding
ACWW 34 4
TIGR 213 14 87
PGIR 15 15
Taiwan 240 8
India 107 8
China 48 3
France 2 4
Korea 19 0
Brazil 2

RGP 1091 23
Total 1795 65 102

The table shows the number of BACs claimed by each group that remain to be finished. This totals 1795 clones. It should be noted that for three groups there are significant differences between these numbers and the information submitted to public databases. In addition it is estimated that another 220 BACs will be identified that fill the remaining gaps. Thus, about 2000 clones remain to be sequenced to finished quality. In fact, the members report finding unsequenced clones for 23 gaps, so 65 physical gaps remain.

Concerted efforts are being made to fill the remaining gaps. The strategy that has had the greatest success is the use of unique end sequences to probe all available libraries. There are now one PAC library, three BAC libraries and two 10 kb insert libraries available for this work. In addition, Rod Wing proposes to prepare a 10X fosmid library with random shear inserts to complement this work.

The IRGSP will have to finish clones at the rate of 100 per month to meet the proposed deadline of December 2004. At this meeting a mechanism was put in place to track sequencing progress. This mechanism takes advantage of the Excel tables prepared by Jianzhong Wu which summarize the sequence-based physical map. They contain all of the BACs in the tiling paths and it is anticipated that about 220 more will be added as gaps are filled. The Excel figures will be made available on the web with the BAC names hypertexted to the GenBank updated database so that the sequence status of each BAC and the progress of each group can be tracked in addition to providing map information. The table entitled "Progress in Genome Sequencing of IRGSP members" on the IRGSP home page is being updated to reflect only tiling path clones. This mechanism will also permit the automated tracking of sequencing progress. It is the responsibility of the IRGSP members to report the names, accession numbers if available, and locations of new tiling path clones to Jianzhong Wu in a timely fashion.

Of the 2000 BACs/PACs mentioned above, there is no current funding for finished quality sequence of about 300 clones, although proposals for this work have been submitted to funding agencies.

The IRGSP will use tracking tools to measure sequencing rates and gap filling on the various chromosomal regions. Further, the IRGSP previously made provision for disregarding chromosomal assignments should work in one region require additional capacity. Members recognize that early decisions for shifting resources are required in order to finish the project in a timely fashion.

Next Meeting

The next IRGSP meeting will be on May 15 . It will be hosted by Bin Han in conjunction with the First International Symposium on Rice Functional Genomics May 12-14 in which a number of IRGSP members will be taking part. Information on the symposium can be found at www.ncgr.ac.cn/ISRFG2003.

Posted by Takuji Sasaki and Ben Burr, 23 February 2003

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