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2nd TOMATO FINISHING WORKSHOP chromosome 9

2nd TOMATO FINISHING WORKSHOP chromosome 9. Wageningen, April 24-25, 2008. CURRENT STATUS OF CHROMOSOME 9. Originally, 142 MARKERS -Most of them in heterocromatin -Very few associated to BACs -Gap of 46cM. New BACs anchored to chr9: -Syngenta data -Filter hybridization

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2nd TOMATO FINISHING WORKSHOP chromosome 9

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  1. 2nd TOMATO FINISHING WORKSHOPchromosome 9 Wageningen, April 24-25, 2008

  2. CURRENT STATUS OF CHROMOSOME 9 Originally, 142 MARKERS -Most of them in heterocromatin -Very few associated to BACs -Gap of 46cM New BACs anchored to chr9: -Syngenta data -Filter hybridization -IL mapping of BACs at ramdom

  3. CURRENT STATUS OF CHROMOSOME 9 12 BACs en HTG 1 33 BACs en HTG 2 20 BACs en HTG 3 34% done COMPLETE 65 BACs IN PROGRESS9 BACs 2 BACs sequencing 7 BACs in finishing pipeline 13 phosmids 12 FPC data 1 seed 5 mapping IL (chr12) 4 extending BACs VERIFYING 35 BACs

  4. SEQUENCING AND FINISHING PIPELINE subcloning puC19 validation BAC puC19 extraction, fragmentation & reparation sequencing(Big-dye terminator, AB3730) 7098781nt sequenced 6040781nt non-redundant ~109 kb/BAC ~15 kb overlap/BAC Phred, Phrap, Consed read pair info whole sequence PCRordirect sequencing electronic digestion contigs

  5. IDENTIFYING NEW BACs • BLASTN against BAC end database • IF no candidates found OR overlaps too large THEN oligo design at BAC ends in order to hybridize filters • FILTER HYBRIDIZATION with markers not associated to BACs • IL MAPPING of BACs at random (200 BACs/chr)?? • FPC data from HindIII and MboI libraries at Sanger Institute • Not very useful: Till now, either our BACs were not in a FPC contig or FPC contigs were too poor to find candidates

  6. NEW SEQUENCING TECHNOLOGIES GS-FLX: 11 BACs were sequenced with 454-Roche technology Elegibility criteria: size, difficulty (repeats, poli-A/T tails…), HTG phase Results (100kb BAC, 15-20x): Goods -No risk of loosing parts during library construction -In general, gaps are small -It resolves A-T poli-tails but only if the coverage is high. Bads -If repeats are present the number of contigs increases substantially -Sequencing stops when AT is repeated several times -If ex. AAATTTTTTTTAAAAA, bases at both sides are not clear unless coverage is high

  7. INSTEAD OF A RECIPE… Some tomato jokes: There were three tomatoes: a mum, a dad and a son. The son lagged behind and fell splat on the floor. His dad yelled to him, “Ketchup, son”. A faucet, lettuce and a tomato were in a race... what happened? The faucet was running, the lettuce was ahead, and the tomato was trying to ketchup! What is a tomato with a cloak? A super-tomato What is a potato with a cloak? A potato with a super-tomato costume

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