sequencing the maize genome maize genome sequencing consortium [email protected]
TRANSCRIPT
Sequencing the Maize Genome
Maize Genome Sequencing [email protected]
Sequencing Progress
Genetic Physical Synteny
A 22 Mb sequence contig on Maize chromosome 4 Maize Chr4
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
Today’s Agenda9:00 – 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
Maize Genome Sequencingat Arizona
Rod A. Wing
Arizona Genomics Institute BIO5
Department of Plant SciencesUniversity of Arizona
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 databaseBAC 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
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rk
10
20
30
40
50
60
70
80
90
100
Pe
rce
nta
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Chromosomes
1 2 3 4 5 6 7 8 9 10
Estimated Chromosomal Coverage
The chromosomal coverage based on maize cv Seneca 60
PhysicalGenetic
•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)
• 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
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
Library Picking
• 60 cycles to look through
1,221 384-well plates for
16,320 clones
BAC End Sequencing(for Clone Verification)
170 96-well plates for
16,320 clones generating
48,960 BES
(2 forward, one reverse)
DNA Preparation and Shearing
170 96-well plates for
16,320 clones
10 plates each month
2.5 plates per person
Genetic Physical Synteny
MegaContig 182 in Maize Genome and Its Synteny to Rice Maize Chr4
26 MB
All ordered and orientated
6.9 Mb(1.5 gap/BAC)
7.2 Mb(1.7 gap/BAC)
Maize Chr9L Rice Chr3S Maize Chr1S
Maize Pseudomolecules for Rice Syntenic Chr3S