PAA / Solanaceae 2006 - July 23-27, 2006 - Madison, Wisconsin, USA Sequencing the gene-rich space of...
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Transcript of PAA / Solanaceae 2006 - July 23-27, 2006 - Madison, Wisconsin, USA Sequencing the gene-rich space of...
PAA / Solanaceae 2006 - July 23-27, 2006 - Madison, Wisconsin, USA
Sequencing the gene-rich space of tomato chromosome 7
Current status of the French effort
Farid RegadGenomic and Biotechnology of Fruit
UMR990 INRA/INP-Toulouse [email protected]
PAA / Solanaceae 2006 - July 23-27, 2006 - Madison, Wisconsin, USA
Chromosome 7 project
Sequencing of gene-rich euchromatic region of chromosome 7– Genetic length: 112 cM– Number of linked markers: 237– Estimated number of BACs to be sequenced: 277
PAA / Solanaceae 2006 - July 23-27, 2006 - Madison, Wisconsin, USA
Chromosome 7 project
Fundings:– INRA, allowing the start of the project– French National Research Agency, starting January
2006– EU-SOL EU-SOL, starting September 2006
PAA / Solanaceae 2006 - July 23-27, 2006 - Madison, Wisconsin, USA
Status of the project
Toulouse sequencing team
Sequencing pipeline
Validation of seed BACs– FISH localisation– IL validation
Sequencing statusShotgun coverage optimized by clone selection
(DACS)
PAA / Solanaceae 2006 - July 23-27, 2006 - Madison, Wisconsin, USA
INRA Toulouse sequencing team
Mondher Bouzayen Project leader
Farid Regad Project management
Seed BAC validationCorinne Delalande BAC selectionPierre Frasse Minimum Tiling Path
Physical mapping
Mohamed Zouine Bioinformatics
http://gbf.ensat.fr
PAA / Solanaceae 2006 - July 23-27, 2006 - Madison, Wisconsin, USA
Farid RegadMohamed Zouine
Corinne Delalande Pierre Frasse
Mondher Bouzayen
Bioinformatics : C. Gaspin (Genopole Toulouse bioinformatic Plateforme)
FISH :O. Coriton (DGAP Rennes)
S. Stack (Colorado, USA) Z. Cheng (Pékin, Chine)
Seed BACs, BAC libraries, genetic maps, IL linesJim Giovannoni, Steve Tanksley (USA), Syngenta, Dani Zamir (Il)
Main investigators
BAC libraries management, BAC filters, hybridizations :Hélène Bergès (CNRGV Toulouse)
PAA / Solanaceae 2006 - July 23-27, 2006 - Madison, Wisconsin, USA
Sequence analysesSequence analyses
GBF GBF
Sequencing pipeline
SequencingSequencing
Genome expressGenome express
AnnotationAnnotation
INRA Toulouse Bioinformatics PlatformINRA Toulouse Bioinformatics Platform
Seed BACs selection Seed BACs selection
Data exchange and storage in local Data exchange and storage in local databasedatabase
NCBI SGN
EUSOL
Location validation on chromosome 7Location validation on chromosome 7
IL: GBFIL: GBF FISH: China / France / USAFISH: China / France / USA
Overlaping BAC selectionOverlaping BAC selection
GBF GBF
PAA / Solanaceae 2006 - July 23-27, 2006 - Madison, Wisconsin, USA
Physical mapping of tomato BACs on chromosome 7
FISH: Fluorescence In-Situ Hybridization
– BAC probes hybridised on pachytene chromosomes or on mitotic chromosomes
IL (D. Zamir)
– BACs mapping on Introgressed Lines (ILs)
PAA / Solanaceae 2006 - July 23-27, 2006 - Madison, Wisconsin, USA
pericentricheterochromatin
TG216
TG438
T1112T1355
T1328
T1428
T1962T1414
T1497
T0676
TM18
CT54
T0966
T0731
TM15
T1347
T1257
T0848
pericentricheterochromatin
309B15
euchromatin
euchromatin
centromere
telomere
telomere
chromomere
euchromatin
059P18130B18
167K07
213E05
230E07
232G04
241F16308M01
309F18
215P04
195N01
Song-Bin Chang Steve Stack
(Colorado USA)
Olivier Coriton (INRA Rennes)
FISH mapping of tomato BACs
PAA / Solanaceae 2006 - July 23-27, 2006 - Madison, Wisconsin, USA
Courtesy of Song-Bin Chang and Steve Stack (Colorado)
PAA / Solanaceae 2006 - July 23-27, 2006 - Madison, Wisconsin, USA
BACs are in the process of FISHing
– 10 BACs: Collaboration with Steve Stack, Colorado, USA (4 assigned on chr. 7)
– 20 BACs: Collaboration with Zhukuan Cheng, China
– Other BACs will be FISHed by HIS platform INRA Rennes, France
FISH mapping status
PAA / Solanaceae 2006 - July 23-27, 2006 - Madison, Wisconsin, USA
Location confirmation - IL
Hba0002M15
PAA / Solanaceae 2006 - July 23-27, 2006 - Madison, Wisconsin, USA
Status of the project
9 BACs shotgun libraries underway17 BACs, PHASE1 done3 BACs, PHASE2 done1 BAC finishedShotgun coverage will be optimized by clone
selection (DACS, patented by Genome-express)
PAA / Solanaceae 2006 - July 23-27, 2006 - Madison, Wisconsin, USA
Sizing
Phrap40
LE_HBa0325D07
LE_HBa0002D20
LE_HBa0095C18
LE_HBa0188B22LE_HBa0215P04
LE_HBa0309B15
LE_HBa0309F18
LE_HBa0033O01
LE_HBa0130B18
LE_HBa0023C09 LE_HBa0166A09
LE_HBa0163O04SL_MboI0031B19
LE_HBa0308M01
LE_HBa0037F23
LE_HBa0059P18
LE_HBa0230E07
LE_HBa0130B18
SL_MboI0119A22
LE_HBa0002M15
LE_HBa0241F16
SL_MboI0017L19
LE_HBa0001N06
40000 60000 80000 100000 120000 140000 160000
40000
60000
80000
100000
120000
Estimations
7,5kb
PAA / Solanaceae 2006 - July 23-27, 2006 - Madison, Wisconsin, USA
Assembly
Validation of Waterman hypothesis– identification of 2 problematic BACs
Coverage (x)
LE_HBa0002D20LE_HBa0095C18
LE_HBa0188B22
LE_HBa0325D07
LE_HBa0215P04
LE_HBa0309B15SL_MboI0031B19
LE_HBa0309F18
LE_HBa0308M01
LE_HBa0033O01
LE_HBa0037F23
LE_HBa0059P18
LE_HBa0230E07
LE_HBa0130B18
SL_MboI0119A22
LE_HBa0002M15 LE_HBa0023C09
LE_HBa0241F16
SL_MboI0017L19
LE_HBa0001N06
LE_HBa0163O04
4 6 8 10 12 14
15
20
25
30
Nunber of contigs normalised for a 100kbp BAC
PAA / Solanaceae 2006 - July 23-27, 2006 - Madison, Wisconsin, USA
Single terminator, dye primer sequencing reaction Pooling of 4 reaction products per capillary
GE-DACS™ Technology
multiplexedsignatures
GENOME express
PAA / Solanaceae 2006 - July 23-27, 2006 - Madison, Wisconsin, USA
GE-DACS™ Technology
Signature-to-sequence comparison– Alignment of pseudo-sequence against expected identity
adaptivethresholding
expectedidentity
pseudosequence
GENOME express
PAA / Solanaceae 2006 - July 23-27, 2006 - Madison, Wisconsin, USA
GE-DACS™ Technology Signature-to-signature comparison
– Segment comparisons by cross-correlation
– ‘Correlogram’ based correspondence detection
correlogram
GENOME express
PAA / Solanaceae 2006 - July 23-27, 2006 - Madison, Wisconsin, USA
BAC data summaryseed Bac overlap overgo markersize ctg# sequencing status PCR validation FISH location
LE_HBa0002D20 TG418 ND DACS Yes US telomereLE_HBa0003E13 ND Yes ChinaLE_HBa0023C09 C2_At2g42810 1130 PHASE I YesLE_HBa0030F21 ND Yes ChinaLE_HBa0033O01 ND COMPLETED YesLE_HBa0034H08 ND Yes ChinaLE_HBa0037F23 ND PHASE I YesLE_HBa0040A11 ND Yes ChinaLE_HBa0049C13 ND Yes ChinaLE_HBa0049P16 LE_HBa0166A09 ND PHASE I YesLE_HBa0059P18 T0966 4361 PHASE I Yes USLE_HBa0095C18 C2_At4g30950 3594 DACS YesLE_HBa0130B18 TG438 981 PHASE I Yes USLE_HBa0138C17 ND Yes ChinaLE_HBa0163O04 T0028 1175 PHASE I Yes US 41% of the long arm from the centromereLE_HBa0166A09 ND PHASE I YesLE_HBa0167K07 T0731 ND Yes US 93.0 cM, sub-telomeric end LE_HBa0188B22 CT135 2000 PHASE II Yes RennesLE_HBa0213E05 T1355 748 Yes ChinaLE_HBa0215P04 ND PHASE I Yes US subtelomeric long armLE_HBa0230E07 T1328 ND PHASE I Yes USLE_HBa0232G04 T1112 4640 Yes RennesLE_HBa0241F16 TG216 ND PHASE I Yes USLE_HBa0242B17 ND PHASE I Yes RennesLE_HBa0308M01 CT54 ND PHASE I Yes USLE_HBa0309B15 T048 2612 PHASE I Yes US subtelomeric long armLE_HBa0309F18 TM15 ND PHASE II Yes RennesLE_HBa0325D07 T1414 977 DACS Yes USLE_HBa0001N06 LE_HBa0309B15 ND PHASE II YesLE_HBa0002M15 C2_At1g19140 2136 PHASE I YesLE_HBa0079F09 ND ND Shotgun YesLE_HBa0175E07 ND Shotgun YesLE_HBa0226J04 ND ND Shotgun Yes
SL_MboI0017L19 LE_HBa0215P04 ND ND Shotgun YesSL_MboI0031B19 LE_HBa0325D07 ND ND Shotgun YesSL_MboI0119A22 LE_HBa0309B15 ND ND Shotgun YesSL_MboI0046H06 ND ND Shotgun YesLE_HBa0226J04 ND ND Shotgun Yes
SL_EcoRI0019G22 ND ND Shotgun YesSL_EcoRI0099J13 LE_HBa0325D07 ND ND Shotgun Yes
PAA / Solanaceae 2006 - July 23-27, 2006 - Madison, Wisconsin, USA
Expected agenda
January 2006– Official start of the project
June 2006– Optimisation and validation of the sequencing pipeline
September 2006– 21 BACs sequenced
January 2007– 70 BAC completed
June 2007– 150 BAC completed
March 2008– 277 BAC completed
December 2008– Chromosome 7 assembly and Finishing completed
2009– Chromosome 7 annotation
PAA / Solanaceae 2006 - July 23-27, 2006 - Madison, Wisconsin, USA
Acknowledgements Jim Giovannoni, Steve Tanksley, Joyce Van Eck
– Mapping data, seed BACs and BAC libraries
Dani Zamir– IL lines
Syngenta– new markers on chromosome 7
Steve Stack, Zhukuan Cheng, Olivier Coriton – BAC FISHing
INRA, French NRA, EU-SOL– Funding support
CNRGV INRA-Toulouse– BAC libraries storage and handling
SGN Consortium (Lukas Mueller, …)– Bioinformatics and n line access to all relevant data for the sequencing