Tomato Chromosome 8 sequencing at Kazusa DNA Research Institute Erika Asamizu.

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Presentation transcript:

Tomato Chromosome 8 sequencing at Kazusa DNA Research Institute Erika Asamizu

Sequencing efforts in Japan Chiba National Institute of Vegetable and Tea Science &

Status of chr.8 sequencing StatusNr. BACs Finished21 ( 2,485,895 bp) Phase 22 Assembling8 Sequencing11 Total42

Status of BAC selection Number of markers selected as seed: 33 Candidate BAC verified: 25 Finished: 18 BAC extension: 15 (Overlap avr. 8 kb) Unable to verify: 8 3D DNA-pool screening

3D DNA pool of BAC library A19 B210 C311 D412 E5P1 F6P2 G7P3 H8P4 Library#384 plate Insert size average (kb) Length (Mb) Genome coverage LE_HBa x SL_MboI x SL_EcoRI x Total964.49x BAC library in 384-plate 1st screening: 384-plate pool in 96-plate 2nd screening: 24 pools of 8 vertical, 12 horizontal and 4 plate pools

FISH localization of ambiguously mapped BAC cLET-5-O24 HBa0076J13 (58 kb) HBa0059L14 (60 kb) cLEB-7-J7 HBa0157P11 (124 kb) MarkerChrcMConfidenceTypeAnchored BAC cLET-5-O24847F(LOD3)ESTHBa0076J13 cLEB-7-J73128IRFLPHBa0059L14 FISH of HBa0157P11 is being performed in Colorado Sate U.

How much to be sequenced? 31,838 TIGR TCs vs. Finished BAC sequences (11.8 Mb) 1375 TCs (4.3%) If 11.8 Mb represent 4.3% of the euchromatic region, entire length would be Mb. (29% of 950 Mb) BLASTN ≥100 bp, ≥97% Still underestimation?

Repeats in gene spaces 30,288 BAC ends vs. 14,229 repeat sequences (TIGR_SolAth_repeat, mips_repeat_collection, SGN repeat collection) 17,412 BAC ends (57.5%) hit repeat sequences. BLASTN E≤1e-10 Euchromatin could be longer because of dispersed type of repeats?

Observed problem HBa0127P15: Anchored to C2_At4g31130 (Verified) Finished length was only 39,599 bp, and did NOT contain the marker sequence. Repeat λHind III 2. BAC/Not I 3. BAC/Xho I 4. 1 kb ladder The digested pattern indicates that the length of sequenced BAC is ~40 kb. Where did the marker go?

Possible problem caused by dispersed type of repeats We found the marker sequence in the shotgun library reads, but it was not incorporated into the finished assembly. Deleted clone was preferentially replicated in host cell? Deletion has occurred because of the repeat?

Possible complementary method in the later stage Extension by fosmid library (Information from J. Van Eck) Methyl filtration 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

Plant genome sequencing team Shusei Sato Erika Asamizu Shigemi Sasamoto Sayaka Shimpo Naoko Torikai Takakazu Kaneko Akiko Watanabe Akiko Muraki Yoshie Kishida Naomi Nakazaki Midori Kato Kumiko Kawashima Yoshimi Shimizu Chiharu Minami Chika Takahashi Kyoko Hayashi Yasukazu Nakamura Manabu Yamada Mitsuyo Kohara Shinobu Nakayama Tsunakazu Fujishiro Mayumi Iriguchi LibrariesSequencing Informatics Satoshi Tabata Hiroyuki Fukuoka Satomi Negoro Yumika Kitamura Molecular Genetics and Physiology team National Institute of Vegetable and Tea Science