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Kazusa DNA Research Institute. Established in 1994 Three departments-6 laboratories 30 researchers, 40 technicians etc. Budget: 19 M$ (Local:Fed = 8:2). Chiba Prefecture. Kazusa DNA Research Institute. Plant Genomics team. Libraries. Sequencing. Informatics. Shusei Sato Erika Asamizu
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Kazusa DNA Research Institute Established in 1994 Three departments-6 laboratories 30 researchers, 40 technicians etc. Budget: 19 M$ (Local:Fed = 8:2) Chiba Prefecture Kazusa DNA Research Institute
Plant Genomics team Libraries Sequencing Informatics Shusei Sato Erika Asamizu Akiko Watanabe Chiaki Kiyokawa Sayaka Shinpo Naoko Torikai Takakazu Kaneko Shigemi Sasamoto Akiko Watanabe Akiko Muraki Mizuho Ishikawa Yoshie Kishida Naomi Nakazaki Midori Kato Kumiko Kawashima Ai Matsuno Yoshimi Shimizu Kyoko Hayashi Yoko Hasegawa Yasukazu Nakamura Tsunakazu Fujishiro Shinobu Nakayama Mitsuyo Kohara Manabu Yamada Mayumi Iriguchi
Microbial genome sequencing project Cyanobacteria Anabaena sp. PCC 7120 6.4 Mb (2001) Gloeobacter violaceus PCC 7421 4.6 Mb (2003) Synechocystis sp. PCC 6803 3.6 Mb (1996) Thermosynechococcus elongatus BP1 2.6 Mb (2002) Rhizobia Mesorhizobium loti MAFF303099 7.6 Mb (2000) Bradyrhizobium japonicum USDA110 9.1 Mb (2002)
A. thaliana genome sequencing project August 1996 - December 2000 55% 25% 20%
Ongoing sequencing projects Eucalyptus camaldulensis 660 Mbp 2004~ Lotus japonicus 470 Mbp 2000~
Procedure Selection of clones to sequence by PCR Confirmation of integrity (Fingerprinting and PCR based on the end sequences) Shotgun libraries (sonication -> pBluescript) Template preparation (TempliPhi) Sequencing (ABI 3700, 3730) x6 coverage Assembly (Phrap) Manual editing and re-sequencing Sequencing of tomato Chromosome 8 Features Genetic length: 87 cM Length of the euchromatic regions: approxomately 17 Mb Number of the markers as seed points: 97 Estimated number of the clones to sequence: 175
Confirmation – integrity and map position 1. Fingerprinting and PCR Selected clone PCR amplification FTC We need the genomic libraries and the end sequence data !
Confirmation – integrity and map position Probe or primers Selected clone FTC 2. Multiple positive (over-go hybridization and PCR) clones in a single FPC
Confirmation – map position Probe or primers FTC FTC 2. Multiple positive (over-go hybridization and PCR) clones in a single FPC
Pipeline for gene modeling and annotation 1. Similarity search 2. ab initio prediction Automatic modeling Manual modeling Low-throughput Higher accuracy High-throughput Annotation