sequencing the maize genome maize genome sequencing consortium rwilson@watson.wustl.edu

Sequencing the Maize Genome

Maize Genome Sequencing Consortiumrwilson@watson.wustl.edu

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

Fra

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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