2 nd TOMATO FINISHING WORKSHOP chromosome 9 Wageningen
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2 nd TOMATO FINISHING WORKSHOP chromosome 9 Wageningen, April 24 -25, 2008 www. sistemasgenomicos. com
CURRENT STATUS OF CHROMOSOME 9 Originally, 142 MARKERS -Most of them in heterocromatin -Very few associated to BACs -Gap of 46 c. M New BACs anchored to chr 9: -Syngenta data -Filter hybridization -IL mapping of BACs at ramdom www. sistemasgenomicos. com
CURRENT STATUS OF CHROMOSOME 9 COMPLETE 65 BACs IN PROGRESS 9 BACs VERIFYING 35 BACs 12 BACs en HTG 1 33 BACs en HTG 2 20 BACs en HTG 3 34% done 2 BACs sequencing 7 BACs in finishing pipeline 13 phosmids 12 FPC data 1 seed 5 mapping IL (chr 12) 4 extending BACs www. sistemasgenomicos. com
SEQUENCING AND FINISHING PIPELINE subcloning pu. C 19 validation BAC pu. C 19 extraction, fragmentation & reparation sequencing (Big-dye terminator, AB 3730) 7098781 nt sequenced 6040781 nt non-redundant ~109 kb/BAC ~15 kb overlap/BAC Phred, Phrap, Consed read pair info electronic digestion whole sequence PCR or direct sequencing contigs www. sistemasgenomicos. com
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 Hind. III and Mbo. I 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 www. sistemasgenomicos. com
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 (100 kb BAC, 15 -20 x): 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. AAATTTTAAAAA, bases at both sides are not clear unless coverage is high www. sistemasgenomicos. com
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 www. sistemasgenomicos. com
