EU-SOL 2008 November 13-16, Toulouse, FRANCE

CHROMOSOME 7 SEQUENCING

Current status and perspective

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

EU-SOL 2008 November 13-16, Toulouse, FRANCE

INTRODUCTION

• Chromosome 7 :

– 27 Mbases of gene-dense euchromatin first estimated

– 25.1 Mb according to Chang et al, 2008

– Initially 270 BACs to be sequenced (new estimation 250 BACs)

• BAC selection:

– Selection and verification of the BACs on chromosome 7 by GBF (INP-T)

– Generation of new tools and resources for rapid selection of the BACs to be sequenced

• BAC sequencing:

– Private Company "Cogenics"

– From draft production to BAC finishing (phase 3)

EU-SOL 2008 November 13-16, Toulouse, FRANCE

Sequencing Strategy

• Until May 2008 :

– BAC sequencing using capillary sequencers

– Finishing strategy :

• Walking on shotgun clones when clone links are available

• Direct BAC sequencing between scaffolds

• From June 2008:

– 454 GS-FLX using Long Paired End Tag reads and

Multiplex Identifiers (MIDs) :

• First batch of 12 BACs sequenced ( 1/2 run)

– 4 BACs directly phase 2 – 1 contig

– 7 BACs phase 2 - 2 to 11 contigs

– 1 problematic BAC with repeats

• results now available on Genbank and SGN (These BAC sequences have been generated with 454 technology)

EU-SOL 2008 November 13-16, Toulouse, FRANCE PAG January 11th, 2009

State of Chromosome 7 progress January 2009

17.7 Mb sequenced of which 14.7 Mb are non redundant (17% of redundant sequences)

60 % of 25 Mbases

19 + 7 FOS phase 0

49 phase 1

84 phase 2

22 phase 3

125 Available

sequences

Selected 174 BACs

7 FOS

Seed BACs 89

Overlapping 85 BACs

7 Fos

EU-SOL 2008 November 13-16, Toulouse, FRANCE PAG January 11th, 2009

Contigs of BACs on chromosome 7

– 163 BACs and FOS are included in 42 contigs on chromosome 7

– 18 BACs remain single

1.13 Mb / 2 cM

1.75 Mb / 22,5 cM

Number of members Number of contigs

19 1

13 1

9 1

7 1

6 1

5 3

4 5

3 16

2 13

0.9 Mb / 0.7 cM

EU-SOL 2008 November 13-16, Toulouse, FRANCE PAG January 11th, 2009

8.654.681.33

0 2.0

1.2

2.0

2.0

4.0

6.0

13.

0

17

18

20

22

22.

3

22.

5

23.

0

23.

5

25.

0

27.

02

8.0

29.

0

31.

03

3.0

35.

03

6.5

38.

0

38.

4

40.

04

4.9

40.

74

2.0

42.

04

2.5

43.

0

44.

34

4.6

46

51

56

63.

06

8.0

72.

5

95.

0

1 Mbase

Centromere

Short arm Euchromatin

Pericentromeric heterochromatin

Long arm Euchromatin

Mbases Sequenced

Mbases Estimation

18.953.96.2

Sequence coverage of chromosome 7

To be sequenced

4.87 10.25

30% of the sequences belong to heterochromatin

EU-SOL 2008 November 13-16, Toulouse, FRANCE

Generation of 3D-DNA pools and Macroarray filters from BAC and Fosmids libraries

These resources were generated in collaboration with the

French Plant Genomic Resource Centre (http://cnrgv.toulouse.inra.fr/)

3D DNA pools

- Half of the HindIII BAC library : 7.8x

- The entire MboI BAC library : 7.5x

Available for the community and already distributed to Spain, Italy, UK, USA, Israel, India, Germany

Macroarray filters- The entire EcoRI BAC library : 8x- Half of the Fosmids library (150000 including 50000 end sequenced) : 6.1x

Available for the community : the filters can be either sent for hybridization or the CNRGV can proceed to the hybridizations

EU-SOL 2008 November 13-16, Toulouse, FRANCE

Details on the Macroarray filters (high density)

• 108 plates 384 spotted on 1 membrane (22 x 22 cm) with duplicated spots on a 6x6 pattern.

• EcoRI BAC library has been spotted on 2 filters • Fosmids library has been spotted on 4 filters

• Image analysis software needed : HDFR3 by INCOGEN

EU-SOL 2008 November 13-16, Toulouse, FRANCE PAG January 11th, 2009

Mb sequenced (NR / R)

13.8 / 18.5

19.5 / 19.8

10.7/212.3

14.7 / 17.7

14.9 6.5 5.1

Location confirmed 96 % 100 % 65 % 100 % 100 % 100 % 100 % 100 %

TGR-2-3 - 4 37% 11% 33% 42% 17% 37% 34% 49% 23% 75% 39% 27%

Selected BACs 35 184 93 178 85 154 165 179 92 17 24 77

Available BACs 19 175 15 154 65 157 125 174 70 4 23 56

TGR2, TGR3 and TGR4 are repeats specific for pericentromeric and centromeric heterochromatin, (Chang et al, 2008)

TGR2, TGR3 and TGR4 sequences against all available BACs (Genbank) to estimate the proportion of heterochromatic BACs for each chromosome

EU-SOL 2008 November 13-16, Toulouse, FRANCE

NextGen Sequencing strategy

• France plan to achieve a 10x coverage by 454 Titanum with Long-PET (starting in February)

• During this we will continue sequencing of batches of BACs and Fosmids by 454

• After achieving the WGS we will sequence more BACs and Fos to fill the gaps on chromosome 7 gene-rich regions

• Need for tight coordination between different inititives (NL, Italy, France, USA, EU6SOL)

• Use of the same DNA sample to be sequenced by all partners ?

PAG January 11th, 2009

EU-SOL 2008 November 13-16, Toulouse, FRANCE

Contributors

GBF (INRA/INP-ENSAT)

Murielle PhilippotPierre FrasseMohamed ZouineFarid RegadMondher Bouzayen

Cogenics CNRGV

Stéphanie Penaud Hélène BergèsHervé Duborjal Sonia VautrinMarcel deLeeuw Elisa PratDiliana DimovaRéjane BeugnotFrançois Pons

Wageningen (NL) Colorado (USA)

Hans De Jong Steve StackDorà Szinay