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2nd TOMATO FINISHING WORKSHOP

chromosome 9Wageningen, April 24-25, 2008

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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

CURRENT STATUS OF CHROMOSOME 9

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COMPLETE 65 BACs

IN PROGRESS

9 BACs

VERIFYING 35 BACs

CURRENT STATUS OF CHROMOSOME 9

2 BACs sequencing 7 BACs in finishing pipeline

13 phosmids12 FPC data 1 seed 5 mapping IL (chr12) 4 extending BACs

12 BACs en HTG 133 BACs en HTG 220 BACs en HTG 3

34% done

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SEQUENCING AND FINISHING PIPELINE

BAC

7098781nt sequenced6040781nt non-redundant

~109 kb/BAC ~15 kb overlap/BAC

validationsubcloning

puC19

extraction, fragmentation &

reparation

puC19

sequencing(Big-dye terminator, AB3730)

contigs

Phred, Phrap, Consed

PCRor

direct sequencing

read pair info

electronic digestion

whole sequence

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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

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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

NEW SEQUENCING TECHNOLOGIES

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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

INSTEAD OF A RECIPE…