FINISHING WORKSHOP APRIL 2008

CHROMOSOME 7THE FRENCH CONTRIBUTION

TG

216

TG

438

T11

12

T13

55

T13

28

T14

28

T19

62T

1414

T14

97

T06

76

TM

18

CT

54

T09

66

T07

31

TM

15

T13

47

T12

57

T08

48

FINISHING WORKSHOP APRIL 2008

INTRODUCTION

• Chromosome 7 :

– 27 Mbases of gene-dense euchromatin

– The coverage of this region is expected to require the sequencing of more than 250 BACs

• BAC selection:

– Selection and verification of the BACs on chromosome 7 by GBF (INPT)

– Generation of new tools and resources allowing the selection and verification of the BACs to be sequenced

• BAC sequencing:

– Private Company Cogenics (Genome Express)

– From draft production to BAC finishing (phase 3)

FINISHING WORKSHOP APRIL 2008

TG

216

TG

438

T11

12

T13

55

T13

28

T14

28

T19

62T

1414

T14

97

T06

76

TM

18

CT

54

T09

66

T07

31

TM

15

T13

47

T12

57

T08

48

Chromosome 7

SELECTION OF BACs

FINISHING WORKSHOP APRIL 2008

BAC selection strategy

• Selection and sequencing of 100 "seed BACs" to cover the gene rich regions of Chromosome 7

• Check the location of each BAC on K7 by FISH and/or by polymorphism screening on ILs

• Selection of overlapping BACs by in silico approaches or 3D DNA pool screening.

FINISHING WORKSHOP APRIL 2008

3D-DNA pools from the BAC libraries

The 3D pools were generated in collaboration with the French Plant Genomic Resource Centre (http://cnrgv.toulouse.inra.fr/)

- Half of the HindIII BAC library: 168 plates 384 64 512 clones 7.8 X genome equivalent

- The entire MboI BAC library : 144 plates 384 52 296 clones 7.5 X genome equivalent

Available for the community

FINISHING WORKSHOP APRIL 2008

Seed BACs selection:

Strategy of Selection Number

of BACs

Overgo assay 20

in silico search using genetic markers (blast against the BES db)

28

Screening of 3D-DNA pools and macroarray filters from the BAC libraries

20

FPC contig : 7

Collaboration / bibliography 12

TOTAL 87

FINISHING WORKSHOP APRIL 2008

Source of genetic markers anchoring the seed BACs:

Sources of markers Number

of BACs

SGN 52

Syngenta 10

SSR Japan 3

Collaboration / bibliography 12

56 markers from Japan on chromosome 7

30 new position (not present in BACs sequenced already) 3 markers have a hit in the BES database 3 markers were screened on the 3D DNA pools 24 more markers available for screening the 3D DNA pools

FINISHING WORKSHOP APRIL 2008

Chromosome 7

STATE OF THE PROGRESS

TG

216

TG

438

T11

12

T13

55

T13

28

T14

28

T19

62T

1414

T14

97

T06

76

TM

18

CT

54

T09

66

T07

31

TM

15

T13

47

T12

57

T08

48

FINISHING WORKSHOP APRIL 2008

Present state of the progress April 2008

14,9 Mb : 55 % of the total estimated euchromatin (27 Mbases) 1,4 Mb of phase 3 sequence

24 in progress

51 phase 1

53 phase 2

21 phase 3

99 Available

sequences

149 Selected

BACs

87 Seed BACs

62 Overlapping

BACs

FINISHING WORKSHOP APRIL 2008

Contigs of BACs on chromosome 7

– 127 BACs are included in 34 contigs on chromosome 7

– Our largest contig is 1.2 Mb long – 28 BACs remain single

number of contigs number of members1 151 122 62 51 4

14 313 2

FINISHING WORKSHOP APRIL 2008

BACs coverage on chromosome 7

Total number of contigs: 34Euchromatin short arm (19 BACs) : 3 contigs and 8 single BACs; 11 GapsEuchromatin long arm (102 BACs): 20 contigs and 11 single BACs; 31 GapsHeterochromatin (28 BACs) : 10 contigs and 4 single BACs

Sequenced BACsBACs under sequencingGap between BACs

0.2 2 3.5 6 15 22 23 28 30 35

39 43 44.5 52 69 78 104 112

Euchro

HeterochromatinEuchro Euchro

Centromere

FINISHING WORKSHOP APRIL 2008

Chromosome 7

BAC SEQUENCING AND FINISHING

TG

216

TG

438

T11

12

T13

55

T13

28

T14

28

T19

62T

1414

T14

97

T06

76

TM

18

CT

54

T09

66

T07

31

TM

15

T13

47

T12

57

T08

48

FINISHING WORKSHOP APRIL 2008

Sequencing Strategy

• BAC sequencing using capillary sequencers

• Finishing strategy : – Walking on shotgun clones when available clone links– Direct BAC sequencing between scaffolds

• 454 Assays on 4 BACs (shotgun + short PETs) : – results available– 1 single scaffold

• 454 assays under progress on 16 BACs (Long PETs)

FINISHING WORKSHOP APRIL 2008

Technical issues

• Empty shotgun subclones : up to 40%

• Repeats : difficulty to obtain a stable assembly

Example : HBa0117J06 (repetitions of more than 5kb conserved at more than 90%)

• Mono- or Di-nucleotide stretches : polymerase fails to pass throught

Example : HBa0076O09 (polyC)

FINISHING WORKSHOP APRIL 2008

The very small BACs

BAC name SGN length final length

SL_EcoRI0071O22 115 11,5

LE_HBa0103N02 149 17,9

LE_HBa0025K11 139 19

LE_HBa0129L05 92 19,7

LE_HBa0073N22 50 28,7

LE_HBa0033O01 47 34,8

LE_HBa0174J08 148 36,7

LE_HBa0189M10 170 45

8 BACs are less than 50kb long.Discrepancy between the size estimated by SGN and the real size found after sequencing (confirmed by restriction digest)

- In some cases we suspect a deletion during clone amplification, then the BAC sequence is not representative of the chromosome sequence - We will sequence other BACs encompassing these areas

FINISHING WORKSHOP APRIL 2008

Other points to be metionned/discussed

Creation of Chromosome 13

We can upload the draft sequences of 4 BACs in Phase 1 or 2 not in chromosome 7

FINISHING WORKSHOP APRIL 2008

People

Genomic and Biotechnology of the Fruits GBF (INRA/INP-ENSAT)

Murielle PhilippotPierre FrasseMohamed ZouineFarid RegadMondher Bouzayen

Cogenics-Genome Express

Stéphanie PenaudHervé DuborjalMarcel deLeeuwDiliana DimovaRéjane BeugnotFrançois Pons