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Sequencing the Maize Genome

Sequencing the Maize Genome. Maize Genome Sequencing Consortium. rwilson@watson.wustl.edu. Sequencing Progress. A 22 Mb sequence contig on Maize chromosome 4. Maize Chr4. Genetic. Physical. Synteny. Plans & Milestones. 22 Mb contig on chromosome 4 Analysis & publication

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Sequencing the Maize Genome

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

  2. Sequencing Progress

  3. A 22 Mb sequence contig on Maize chromosome 4 Maize Chr4 Genetic Physical Synteny

  4. 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

  5. Today’s Agenda 9:00 – 9:15 am Introductions and Project Overview (Rick Wilson) 9:15 – 10:30 am Progress 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 am Break 10:45 – Noon Progress Updates-continued - Assembly Improvement (Pat Schnable) - Annotation and Data Display (Doreen Ware) - DOE Project (Dan Rohksar) - Outreach (Dick McCombie, Doreen Ware) Noon – 1:00 pm Executive Session-Working Lunch 1:00 – 2:00 pm Discussion 2:00 pm Depart for Airport

  6. Maize Genome Sequencingat Arizona Rod A. Wing Arizona Genomics Institute BIO5 Department of Plant Sciences University of Arizona

  7. 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) Genetic Anchoring in silico, overgo hybridization (19,292) Framework Fingerprinting ~460,000 BACs BAC End Sequencing ~800,000 BAC physical maps (HICF & Agarose) FPC databases (Agarose and HICF) STC database Choose a seed BAC (800 Kb spacing) Shotgun sequencing and finishing STC database search, FP comparison Determine minimum overlap BACs Complete maize genome sequence

  8. Estimated Chromosomal Coverage 100 Physical 90 Genetic 80 70 60 Percentage 50 40 30 20 10 1 2 3 4 5 6 7 8 9 10 Chromosomes The chromosomal coverage based on maize cv Seneca 60

  9. Minimum Tiling Path Pipeline(CSHL/AGI) • 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.

  10. Clone Picking Progress • 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

  11. 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

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

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

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

  15. MegaContig 182 in Maize Genome and Its Synteny to Rice Maize Chr4 All ordered and orientated 26 MB Genetic Physical Synteny

  16. Maize Pseudomolecules for Rice Syntenic Chr3S 6.9 Mb (1.5 gap/BAC) 7.2 Mb (1.7 gap/BAC) Maize Chr9L Rice Chr3S Maize Chr1S

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