chromosome 8 sequencing: current status and future prospects toward finishing shusei sato, erika...

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