EU-SOL 2008 November 13-16, Toulouse, FRANCE CHROMOSOME 7 SEQUENCING Current status and perspective TG216 TG438 T1112 T1355 T1328 T1428 T1962 T1414 T1497.

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EU-SOL 2008 November 13-16, Toulouse, FRANCE CHROMOSOME 7 SEQUENCING Current status and perspective TG216 TG438 T1112 T1355 T1328 T1428 T1962 T1414 T1497 T0676 TM18 CT54 T0966 T0731 TM15 T1347 T1257 T0848

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 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, FRANCEPAG January 11th, 2009 State of Chromosome 7 progress January Mb sequenced of which 14.7 Mb are non redundant (17% of redundant sequences) 60 % of 25 Mbases

EU-SOL 2008 November 13-16, Toulouse, FRANCEPAG 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 membersNumber of contigs Mb / 0.7 cM

EU-SOL 2008 November 13-16, Toulouse, FRANCEPAG January 11th, Mbase Centromere Short arm Euchromatin Pericentromeric heterochromatin Long arm Euchromatin Mbases Sequenced Mbases Estimation Sequence coverage of chromosome 7 To be sequenced % 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 ( 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 ( including 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, FRANCEPAG January 11th, 2009 Mb sequenced (NR / R) 13.8 / / / / Location confirmed96 %100 %65 %100 % TGR %11%33%42%17%37%34%49%23%75%39%27% Selected BACs Available BACs 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 Philippot Pierre Frasse Mohamed Zouine Farid Regad Mondher Bouzayen CogenicsCNRGV Stéphanie Penaud Hélène Bergès Hervé Duborjal Sonia Vautrin Marcel deLeeuwElisa Prat Diliana Dimova Réjane Beugnot François Pons Wageningen (NL) Colorado (USA) Hans De Jong Steve Stack Dorà Szinay