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Minutes of the Technical Workshop
Tsukuba, Japan
8 June, 2001
Strategy by Group
Each PI gave an estimate of the amount of sequence (phase 2 + complete) that could be accomplished in the next calendar year, and reported on strategy they would employ.
| Chromosome | Current* Completed Mb |
Next Year Mb |
Estimated Size Mb |
Strategy next year |
| 1 | 47 | 1 | 52 | phase 2 |
| 2 | 0.1 | - | 44 | - |
| 3 | 7 | 35 | 46 | BAC by BAC finishing |
| 4 | 4 | 30 | 36 | phase 2 followed by finishing |
| 5 | 1 | 20 | 34 | phase 2 |
| 6 | 13 | 21 | 35 | phase 2 |
| 7 | 4 | 28 | 33 | phase 2 |
| 8 | 0 | 30 | 34 | phase 2 |
| 9 | 0.5 | 1-2 | 26 | phase2 |
| 10 | 13 | 10-11 | 23 | BAC by BAC finishing |
| 11 | 0.15 | 5-10 | 34 | BAC by BAC finishing + phase 2 |
| 12 | 1 | 28 | 30 | BAC by BAC finishing |
| Total | 91 | 209-216 | 427 |
*Finished and Phase 2
Some of the highlights:
Bin Han reported that his group would finish sequencing chromosome 4 by January 2002, both in the indica strain, Guangluai4, and in Nipponbare at least in the phase 2 level. These sequences will be taken to completion. China will be looking to sequence in another chromosoal region in 2002.
Robin Buell reported that chromosome 10 would be completed at the finished level by January 2002. TIGR has all BACs for the entire bottom arm of chromosome 10 in production, with the exception of a 10 kbp and a 30 kbp gap. TIGR is currently working on 1-2 Mb on the top arm of chromosome 10 as well. On chromosome 3, TIGR has 10 Mb in production and has 17 Mb remaining on their allocation. TIGR anticipates 10 of this 17 Mb will be readily put into production by early 2002. The remaining 7 Mb will be of BAC clones that are difficult to identify and will require additional efforts to complete. TIGR is currently requesting an additional allocation on either chromosome 3 and 11.
Ho-Il Kim reported his group had completed 5 PACs and 8 BACs on chromosome 1 and were now expecting to sequence a region on chromosome 9 at the rate of 1-2 Mb a year. Two of BACs can be sequenced monthly with phase 2 level. Therefore he would like to start he sequencing from 77.3cM region on chromosome 9. Within this 1700kb region the RGP has verified 3 Monsanto contigs.
Takashi Matsumoto reported that the RGP would sequence 100 Mb at the phase 2 level and 20 Mb at the finished level in their 2001 fiscal year beginning April, 2001.
Francis Quetier reported that France expected to complete a tiling path for all of chromosome 12 this fall. Template preparation will be completed in December and full scale sequencing would begin in January. He expected that Genoscope would complete chromosome 12 at the finished level by June 2001.
Francis reported that the bottleneck was access to the raw data, specifically, raw fingerprint data.
Teh-Yuan Chow reported that Taiwan had completed 14 PACs on chromosome 5. He expects that they will be able to sequence 200 BACs in the coming year. This very large increase is the result of increased capacity that he hopes to take advantage of in January 2002.
Rod Wing reported that CCW will have 11 Mb each completed at the finished level both on chromosome 10 and on chromosome 3 by this time next year.
Jo Messing reported that the Rutgers group had completed a 21 BAC contig, about 3 Mb on chromosome 10, mostly at the finished level, and was now sequencing on chromosome 11. They have picked a region between 28 and 30.3 cM in the Adh1-Adh2 region, and expect to complete 3 Mb at the phase 2 level by this time next year.
Jo also reported that India has selected a nucleation point at 97.4 cM on chromosome 11.
Physical Mapping
Jianzhong Wu reported on the RGP strategy for preparing a physical map. He has just finished assigning 6,600 ESTs to the YAC physical map. This increased the YAC coverage to 80% of the genome. The RFLP map now contains 3267 markers and covers 1530 cM. 2000 of the markers are ESTs and a total of 2500 of the genetic markers contain rice sequence. Both the sequenced markers and the mapped ESTs were used to BLAST the sequenced PACs and the Monsanto BACs.
On chromosome 1 the RGP has completed 41.2 Mb of finished and phase 2 sequence (approximately 81% according to current estimates of the size of the chromosome). There are 11 gaps for which he needs to estimate the sizes. Of the 1054 markers on this chromosome, 99% hit sequenced BACs or PACs.
His group anchored sequenced Monsanto BACs by BLASTing the entire 3,300 set against markers followed by PCR confirmation. Totals they assigned to the 12 chromosomes are:
413, 285, 319, 203, 191, 177, 210, 168, 108, 152, 127, 128, Total = 2481
Of these, 211 had discrepancies relative to the Monsanto assignments.
Wu reported developing minimum tiling paths on chromosomes 7 and 8 by anchoring Monsanto BACs with RGP markers:
chromosome 7 157 BACs
chromosome 8 112 BACs
In a further step his group screened RGP PAC/BAC libraries to fill gaps in Monsanto contigs. They used RGP STS and ESTs markers that fell within gaps of Monsanto BAC contigs followed by PCR confirmation. The next step will be to use CUGI STCs and fingerprints to find further gap filling BACs. Finally, they will do end walking. These four steps will be completed by December at which time, Dr. Wu expects to have 70-90% coverage.
Rod Wing offered to fingerprint the 1000 PACs that had so far been placed in the gaps to look for overlaps with their clones.
Dick pointed out that the Monsanto BACs had 15-30% overlap, a characteristic of the library, and that it would be wasteful to rely on them exclusively to build a tiling path.
Rod Wing summarized the current results from the joint efforts of his group and Monsanto to integrate the sequenced Monsanto BACs into the physical map. His presentation can be viewed at Rod Wing's Slides. [Don't be discouraged by the first blank slide.]
The information on the integration of the Monsanto and CUGI clones can be found at http://www.genome.clemson.edu/projects/rice/fpc/ which will take you to WebFPC.
Sequencing Strategy at the RGP
Yuichi Katayose talked about template preparation at the RGP. They have taken advantage of an impressive number of innovations and are using automated steps to improve capacity. They have achieved template preparation at a cost of $50/96-well plate.
Kimiko Yamamoto talked about the RGP sequencing strategy. First of all, she talked about the number of BACs that were subjected to 10X coverage and were phase 2 quality after automated assembly. When the RGP did 10X coverage on an unsequenced BACs, they obtained 85% phase 2 quality. When they took the nominal 5X Monsanto coverage and added another 5X coverage of their own, they obtained 80% in phase 2 after assembly. Those phase 1 clones that failed to give phase 2 quality are sent directly to finishing. Software developed by Dr. Sakata's group for phase evaluation is in the testing stage. They found that in its current state it had about 65% agreement compared with manual classification.
She reported that the RGP now has 25 3700 ABI sequencers and has a capacity of 10 Mb/month or 100 BACs/month with 10X coverage and 200 BACs/month doing only 5X coverage on Monsanto BACs.
Their has risen to 60 phase 2 BACs released per month and their finished clones have remained constant at 10 PACs/BACs per month. The RGP has increased the number of finishers to 10, and has improved efficiency by assigning difficult BACs to the most qualified finishers.
Dick McCombie pointed out that phase 2 sequence may contain errors if the phase assignment was automated. Errors could include bacterial transposons, vector sequences, and parts of more than one project assembled making the assembly a different size from the BAC.
The Hybrid Approach (Multiple sequencing strategies within the IRGSP)
Dick McCombie gave a cogent presentation of his views of current progress and how to finish the genome. He said that the current hybrid approach - where much of the genome was being drafted at phase 2 level while some groups were finishing as they go - had advantages and disadvantages. He pointed out how the IRGSP could take advantage of the current situation to make finishing the genome more feasible. His presentation and recommendations for immediate action can be found at Dick McCombie's Slides.
Dick McCombie also presented his ideas on finishing previously sequenced clones. They are currently testing the idea of making three libraries: Two1-3 kb insert libraries, one each in M13 and a plasmid vector and one 3-5 kb insert library in a plasmid. After shotgun sequencing at low coverage, bridge clones are selected and completely sequence by transposon mutagenesis.
Lincoln Stein presented some lessons from annotation of two finished genomes. His presentation can be found at Lincoln Stein's Slides.
Format of the next meeting
The participants thought highly of the structure of this meeting and we considered how we could replicate it at TIGR (all day September 19). The meeting will be open to the public but we can retain intimate interaction among the principles by having the first two hours in an auditorium with general presentations by each group. The remainder of the time can be spent on specific agenda items at round table in a room that seats 60-70. If extra time is needed, we can have specialty workshops the night before or the evening following the meeting.
Meeting Participants
Dr. Takuji Sasaki, RGP, Japan
Dr. Ben Burr, Brookhaven National Laboratory, USA
Dr. Bin Han, National Center for Gene Research, China
Dr. Francis Quetier, Genoscope, France
Dr. Richard McCombie, Cold Spring Harbor Laboratory, USA
Dr. Lincoln Stein, Cold Spring Harbor Laboratory, USA
Dr. Joachim Messing, Rutgers University, USA
Dr. Robin Buell, The Institute for Genomic Research, USA
Dr. Rod Wing, Clemson Univ. Genomics Institute, USA
Dr. Teh-Yuan Chow, Academia Sinica, Taiwan
Dr. Kim Ho-Il, RDA, Korea
Also from the RGP:
Dr.Takashi Matsumoto
Dr.Katsumi Sakata
Dr.Kimiko Yamamoto
Dr.Jianzhong Wu
Dr.Yuichi Katayose
Dr.Baltazar Antonio
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