Transcript ppt - Sol Genomics Network

Expanding the Tool Kit for BAC
Extension
Summary of completion criteria
developed for NSF
Tomato Sequencing Workshop
January 14, 2007
Resources for Additional Anchor
BACs
• 1) Mapping of random low copy BACs to create a
pool of new seed BACs that could serve all 12
chromosomes.
- low copy BACs identified by the group of Tabata
et al.
- BAC end sequences mapped to S. lycopersicum X
S. pennellii ILs.
-
India, Korea, others
• 2) Sequencing of 2 million plus reads
derived from low copy BACs defined by
Dr. Tabata et al.
• 3) identification of low copy cosmids (US)
that would also be end sequenced by Japan.
• 3) BAC OVERGO screen of MboI library
–
–
–
–
Large inserts 130 kb avg.
FPC data generated by Sanger
10 - 20 probes per country
Screening in next several months
4) Industry
anchored BACs
Goals of the International Tomato
Genome Sequencing Project
Estimate of tomato euchromatin and heterochromatin genome fractions
Based on 50 independent measurements of stained tomato chromosomes
Relative chromosome length
Relative bivalent diameter
Relative area
Relative optical density
Relative OD X relative area
Total OD X area
Fraction of genome
Heterochromatin Euchromatin
0.36
0.64
X 1.23
X 1.00
0.44
0.64
X 4.78
X 1.00
2.10
0.64
/ 2.74
/ 2.74
0.77
0.23
Approximately 23% of the tomato genome
is in the form of euchromatin
Mb size of tomato euchromatin based
on cytogenetic measurements
0.95 pg / tomato genome X 0.23 (euchromatin fraction) =
0.22 pg
965 X 106 pb/pg = 2.12 x 108 bp or 212 Mb
(705 Mb heterochromatin)
Estimate of tomato euchromatin size based on
available EST and genome sequence
15.5 Mb available sequence (Fall 2006)
8,097 high quality unigene set
- all available full-length tomato genes in GENBANK
- TIGR full-length cDNA sequences (redundantly sequenced)
- SGN unigene contigs with 5 or more ESTs
- redundnacy correction
456 of 8,097 genes found in available genome sequence (5.6%)
Correcting for 85% expectation yields 6.6% of target gene space
15.5/0.066 = 239 Mb tomato euchromatin target
Estimate of gene space missed in this approach:
Genes missed in centromere (rice chromosome 8 - 86 genes)
12 x 86 = 1032 centromere genes
Exelisis heterochromatin BACs - 2 BACs representing 200 kb
were sequenced and one gene identified.
705,000 kb in herterochromatin (slide 2)
705,000 / 200 = 3525 heterochromatin genes
35,000 estimated tomato genes - 1032 - 3525 = 30,500 genes
(87%)
Correcting for 3% euchromatin gaps (as in rice) results in 85% of
total tomato gene space is anticipated to be recovered under the
International Tomato Genome Sequencing Project.
Sequencing standards
A “finished BAC” is defined as……
• it contains an error rate of less than 1:10,000 bases and
continuous sequence across the entire BAC (HTGS phase 3)
• has an average of 8-fold redundancy in sequencing coverage
with a minimum of one high quality read in both directions at any
specific sequence
• all reasonable state of the art approaches available at the time
for gap filling will be used
Tomato euchromatin completion criteria:
1) complete sequencing of the major euchromatin “arms” flanking
each of the 12 tomato chromosomes
2) to a degree of completion comparable to the standards of
completion used to guide the international rice genome
sequencing project (IRGSP, 2005) ---- e.g. anticipate 4 - 6 gaps
per chromosome.
Furthermore:
1) Sequence to at least the closest mapped marker to the euchromatin /
heterochromatin border .
2) Attempt to walk until characteristic heterochromatin repeats are
identified and at minimum define the size of the remaining gap
In summary, the target of the
international genome sequencing
effort is sequencing of the euchromatin
arms of all twelve tomato
chromosomes which we estimate
will represent approximately 85%
of the tomato gene space.