2nd tomato finishing workshop chromosome 9
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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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2nd tomato finishing workshop chromosome 9

2nd TOMATO FINISHING WORKSHOPchromosome 9

Wageningen, April 24-25, 2008


2nd tomato finishing workshop chromosome 9

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


2nd tomato finishing workshop chromosome 9

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


2nd tomato finishing workshop chromosome 9

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


2nd tomato finishing workshop chromosome 9

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


2nd tomato finishing workshop chromosome 9

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


2nd tomato finishing workshop chromosome 9

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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