2. Sequencing the Maize Genome Maize Genome Sequencing Consortium rwilson@watson.wustl.edu

3. Sequencing Progress

4. GeneticPhysicalSynteny A 22 Mb sequence contig on Maize chromosome 4 Maize Chr4

5. Plans & Milestones 22 Mb contig on chromosome 4 - Analysis & publication Draft sequence of the maize genome - All BACs: shotgun & pre-finishing (?) - End of the calendar year - Announce at the Maize Meeting in D.C. Completion of the maize genome sequence - Version 1.0 - Analysis & Publication Future Work - Secondary Annotation - Clean-up sequencing, maintenance

6. Todays Agenda 9:00 – 9:15 amIntroductions and Project Overview (Rick Wilson) 9:15 – 10:30 amProgress Updates – WU/AGI/CSHL/ISU Project - Map and Tile Path (Rod Wing) - Library Construction and Production (Lucinda Fulton) - Sequence Improvement (Bob Fulton, Dick McCombie, Rod Wing) - Data Submission (Joanne Nelson) 10:30 - 10:45 amBreak 10:45 – NoonProgress Updates-continued - Assembly Improvement (Pat Schnable) - Annotation and Data Display (Doreen Ware) - DOE Project (Dan Rohksar) - Outreach (Dick McCombie, Doreen Ware) Noon – 1:00 pmExecutive Session-Working Lunch 1:00 – 2:00 pmDiscussion 2:00 pm Depart for Airport

7. Maize Genome Sequencing at Arizona Rod A. Wing Arizona Genomics Institute BIO5 Department of Plant Sciences University of Arizona

8. BAC by BAC Strategy to Sequence the Maize Genome Maize B73 Genome (2300 Mb) BAC library construction (Hind III, EcoR I, MboI ; 27X genome coverage (~150kb inserts) BAC End Sequencing ~800,000 Genetic Anchoring in silico, overgo hybridization (19,292) Fingerprinting ~460,000 BACs STC database BAC physical maps (HICF & Agarose) FPC databases (Agarose and HICF) Choose a seed BAC (800 Kb spacing) Shotgun sequencing and finishing STC database search, FP comparison Determine minimum overlap BACs Complete maize genome sequence Framework

9. 10 20 30 40 50 60 70 80 90 100 Percentage Chromosomes 12345678910 Estimated Chromosomal Coverage The chromosomal coverage based on maize cv Seneca 60 Physical Genetic

10. BAC End Sequence of potential BACs are BLASTed against the Seed BACs Results are classified based on location on the physical map A table for each BAC is created of filtered BLAST results with links to CMap and GBrowse Blast results are imported into CMap and GBrowse with additional information such as trace files and FPCs A table of alignments between the seed BAC and the BAC end sequences contains links to CMap and GBrowse. CMap displays the FPC data for the seed BAC and the candidate BACs to pick. GBrowse provides an alignment of the BES with the seed sequence and displays the trace data. Minimum Tiling Path Pipeline (CSHL/AGI)

11. Seed BACs: 3,400, complete Clone Walking from Seed BACs: 12,824 complete Total clones picked = 16,224 (169 96-well plates) 15,400 successful 7,800 Year 1 7,600 Year 2 Gap-filling ~600 Year 3, in progress Clone Picking Progress

12. Clone Picking Clone Walking –By sequence if seed BAC sequence was available –By fingerprints when no sequence was available Clone verification –BAC end sequence –Seed BAC sequence

13. Library Picking 60 cycles to look through 1,221 384-well plates for 16,320 clones

14. BAC End Sequencing (for Clone Verification ) 170 96-well plates for 16,320 clones generating 48,960 BES (2 forward, one reverse)

15. DNA Preparation and Shearing 170 96-well plates for 16,320 clones 10 plates each month 2.5 plates per person

16. GeneticPhysicalSynteny MegaContig 182 in Maize Genome and Its Synteny to Rice Maize Chr4 26 MB All ordered and orientated

17. 6.9 Mb (1.5 gap/BAC) 7.2 Mb (1.7 gap/BAC) Maize Chr9LRice Chr3SMaize Chr1S Maize Pseudomolecules for Rice Syntenic Chr3S