chromosome 8 sequencing: current status and future prospects toward finishing shusei sato, erika...
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Chromosome 8 Chromosome 8 Sequencing: Current Sequencing: Current
Status and Future Status and Future Prospects toward Prospects toward
FinishingFinishing
Shusei Sato, Erika Asamizu, Takakazu Kaneko, Hiroyuki Fukuoka, Satoshi Tabata
Second Tomato Finishing Workshop, Apr. 24-25, 2008
921651.8
791431.8
671712.6
621372.2
401193.0
631011.6
511122.2
33872.6
401162.9
41872.1
431032.4
391203.1
# anchorscM chr lengthcM per anchor
Distribution of Anchor Markers on Chromosomes
Initial seeds on Chr.8
Sequence strategyWe have been taking the same strategy
applied in the Lotus japonicus genome project.
<Shotgun sequencing of BAC clones>
•vector for the shotgun clone: pUC118•insert size of the shotgun clone: ca. 3 kb•template DNA preparation: TempliPhi•sequencing chemistry: AB Big Dye Terminator
•sequencer: AB 3730•gap closing in finishing phase: 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
2,627 SSR
522 have already been mapped
2,105 new EST SSR
MiBASE(http://www.kazusa.or.jp/jsol/microtom/indexj.html)
EST unigenes (26,363)Full-length cDNA (57,422)
Summary of EST-SSR Marker analysis
712 markers have been mapped on EXPEN2000
chr1 78 chr7 56chr2 66 chr8 62chr3 74 chr9 52chr4 60 chr10 49chr5 62 chr11 50chr6 52 chr12 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
C08HBa0202N15: Presence of highly similar repeat sequences
C08SLm0144I10: Presence of a long (AT) cluster
C08SLe0126A12: Presence of highly similar repeat sequences
C08HBa0045I24: Presence of a long (C) cluster
Gap Filling by Whole Genome Shotgun Sequencing
• Selected BAC Mixture (SBM) shotgun1. Select BACs whose end sequences do not contain un
desired (repeat) sequences2. Mix the BACs and sequence by shotgun
BAC
Repeat Gene space
BLASTN BES vs. RepeatDB
402,012 end sequences from 177,408 BAC clonesvs.
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
Source of selected BAC mixture
LibraryBoth ends are
repeatOne end is
repeatBoth 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
Selected BAC Mixture (SBM) shotgun sequencing
20,000 BAC clones
Six-times the euchromatin coverage
10,000 clones from HBa library 5,000 clones from EcoRI library 5,000 clones from MboI library
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 Dept. Plant Genome ResearchGenome Research
Shigemi SasamotoShigemi Sasamoto
Tsuyuko WadaTsuyuko Wada
Ai MatsunoAi Matsuno
Takakazu KanekoTakakazu Kaneko
Akiko WatanabeAkiko Watanabe
Akiko OnoAkiko Ono
Naomi NakazakiNaomi Nakazaki
Midori Kato Midori Kato
Kumiko KawashimaKumiko Kawashima
Yoshimi Shimizu Yoshimi Shimizu
Chiharu MinamiChiharu Minami
Chika TakahashiChika Takahashi
MarkerMarkerSequencingSequencing
Satoshi TabataSatoshi TabataErika AsamizuErika AsamizuShusei SatoShusei Sato
Hiroyuki FukuokaHiroyuki Fukuoka
Satomi NegoroSatomi NegoroYumika KitamuraYumika Kitamura
Molecular Genetics and Molecular Genetics and Physiology teamPhysiology team
National Institute of Vegetable and Tea Science