Second Tomato Finishing Workshop, Apr. 24-25, 2008
Chromosome 8 Sequencing:
Current Status and Future
Prospects toward Finishing
Shusei Sato, Erika Asamizu, Takakazu Kaneko,
Hiroyuki Fukuoka, Satoshi Tabata
Distribution of Anchor Markers on Chromosomes
Initial seeds on Chr.8
92
165
1.8
79
67
143 171
1.8 2.6
62
137
2.2
40
119
3.0
63
101
1.6
51
112
2.2
33
87
2.6
40
116
2.9
41
87
2.1
43
103
2.4
39 # anchors
120 cM chr length
3.1 cM per anchor
Sequence strategy
We have been taking the same strategy
applied in the Lotus japonicus genome project.
<Shotgun sequencing of BAC clones>
•vector for the shotgun clone:
•insert size of the shotgun clone:
pUC118
ca. 3 kb
•template DNA preparation:
•sequencing chemistry:
•sequencer:
TempliPhi
•gap closing in finishing phase:
AB Big Dye Terminator
AB 3730
primer walking
shotgun clone or BAC direct
<Walking from seed clones>
• BAC end sequence database
Problem
• It is impossible to continue walking from the
small number of seed points
– Extension terminated at 18/33 seed
points
Complementary Efforts in Japan
1. Development of EST-derived new
microsatellite markers to obtain more
seed points for sequencing
2. Gap filling by an alternative sequencing
strategy
Development of New Microsatellite Markers
MiBASE(http://www.kazusa.or.jp/jsol/microtom/indexj.html)
EST unigenes (26,363)
Full-length cDNA (57,422)
2,627 SSR
522 have already been mapped
2,105 new EST SSR
Summary of EST-SSR Marker analysis
712 markers have been mapped on EXPEN2000
chr1
chr2
chr3
chr4
chr5
chr6
78
66
74
60
62
52
chr7
chr8
chr9
chr10
chr11
chr12
56
62
52
49
50
51
34 new seed clones have been selected
Status of Chr.8 Sequencing
Status
Nr. BACs
Finished
128
Phase 2
5
Assembling
12
Sequencing
22
Total
167
Finished length without overlap: 12,562,802 bp
67 seed points (42 contigs, 13 single clones)
Troubled clones
Clones finished in phase 2
C08HBa0050P21:
Presence of a long (AT) cluster
C08SLm0144I10:
Presence of a long (AT) cluster
C08HBa0045I24:
Presence of a long (C) cluster
C08HBa0202N15:
Presence of highly similar repeat sequences
C08SLe0126A12:
Presence of highly similar repeat sequences
Gap Filling by Whole Genome
Shotgun Sequencing
•
Selected BAC Mixture (SBM) shotgun
1. Select BACs whose end sequences do not contain
undesired (repeat) sequences
2. Mix the BACs and sequence by shotgun
BAC
Repeat
Gene space
BLASTN BES vs. RepeatDB
402,012 end sequences from 177,408 BAC clones
vs.
14,229 repeat sequences (TIGR_SolAth_repeat,
mips_repeat_collection, SGN repeat collection)
Percentage
LTR
49.7
Unknown
30.3
rRNA
7.4
Satellite
1.6
Simple repeat
1.5
DNA
1.4
LINE
1.3
Library
Both ends are
repeat
One end is
repeat
Both ends are
NOT repeat
HBa
19,123
35,277
26,181
Eco RI
9,533
18,594
15,516
Mbo I
15,570
20,995
13,134
Total
44,226
74,866
54,831
Source of selected BAC mixture
Selected BAC Mixture (SBM)
shotgun sequencing
10,000 clones from HBa library
5,000 clones from EcoRI library
5,000 clones from MboI library
20,000 BAC clones
Six-times the euchromatin coverage
Status of SBM Shotgun Sequences
・Sequencing started in Feb. 2007.
・As of April 2007, 2.2 million sequences
has been accumulated.
(total length is 1.5 Gbp)
・Assembled into 193,330 contigs.
Total size of the contigs is ~ 484 Mbp.
longest contig: 17,702 bp,
> 5kb contigs: 21,230
Dept. Plant Genome
Research
Satoshi Tabata
Erika Asamizu
Shusei Sato
Sequencing
Shigemi Sasamoto
Akiko Watanabe
Tsuyuko Wada
Akiko Ono
Ai Matsuno
Midori Kato
Kumiko Kawashima
Yoshimi Shimizu
Chiharu Minami
Chika Takahashi
Molecular Genetics and
Physiology team
Marker
Takakazu Kaneko
Naomi Nakazaki
National Institute of Vegetable and Tea Science
Hiroyuki Fukuoka
Satomi Negoro
Yumika Kitamura