International Mammalian Genome Society


The 13th International Mouse Genome Conference
October 31-November 3, 1999

Table of Contents * Structure * Bioinformatics * Sequence * Mapping * New Tools * Gene Discovery * Developmental * Mutagenesis * Functional Genomics

D19 The Mouse Full-Length cDNA Encyclopedia

Jun Kawai1,2, Yoshifumi Fukunishi1,2, Piero Carninci1, Yuichi Sugahara1,2, Masayasu Yoshino1, Yasuhiro Ozawa1, Kazuhiro Shibata1,2, Masayoshi Itoh1,2, Yuko Shibata1,3, Hideaki Konno1,2, Toshinori Endo1, Masami Muramatsu1,2, Yasushi Okazaki1, and Yoshihide Hayashizaki1,2. 1Laboratory for Genome Exploration Research Project, Genomic Sciences Center (GSC) and Genome Science Laboratory, Tsukuba Life Science Center, the Institute of Physical and Chemical Research (RIKEN); 2CREST, Japan Science and Technology Corporation (JST); 3Research and Development, Nippon Gene Co., Ltd.

With the production of the mouse full-length cDNA encyclopedia we are aiming at isolating and sequencing the majority of the 100,000 mouse full-length cDNAs. To meet this target, in recent years we have been developing original technology for full-length cDNA cloning (cap trapper and trehalose-thermoactivated reverse transcriptase) and high-throughput sequencing. In the first phase, representative full-length cDNA libraries are sequenced from the 3' end by RISA384-capillary sequencing instrument, which is coupled to our high-throughput plasmid preparation instrument. To increase the chance to discover new and rare genes, full-length cDNA libraries are normalized and subtracted using a new vector. We also developed a new vector to prepare cDNA libraries of size that reflect the size of the starting mRNA. Until middle of July 99, approximately 42,000 different full-length cDNAs have been selected in more than 276,000 successful sequences from about 90 cDNA libraries. Additionally, libraries receive a score for the quality of full-length by 5' end sequences. In best libraries more than 95% of clones are full-coding. We have produced about 24,500 reads from 5' ends that produced about 13,500 clusters.

Subsequently, non-redundant clones are selected from best scoring libraries and subjected to full-length sequence in the second phase. In this second phase, we give high priorities to the novel cDNA clones and are producing their rough draft sequence. Even though they would contain some low-quality base-calling, whole view of mouse transcriptome sequences must be very valuable for the future analysis. We are applying three strategies for full-length sequencing depending the length of each cDNA clones. 1) Long-read from the both ends for short clones (less than 1.5kb). 2) Primer walking strategy for middle size clones (1.5 - 2.5kb) 3) Shotgun sequence for long clones (more than 2.5kb). At the assembling step, we also utilize the public EST data to fill gaps of cDNA sequences, which can accelerate the proceeding of the full-length sequence project. At this time, we have got the more than 6,000 full-sequence. We believe our encyclopedia will make a great contribution not only to mouse society but also to any fields of life sciences.

We thank Yumi Kojima, Ayako Hara, Mika Yagame, Hirokatsu Suzuki, Shiroh Fukuda, Fumi Hori, Takahiro Matsuyama, Noriko Kikuchi, Saiko Akahira, Norihito Hayatsu, Kenjiroh Sato, Toshiyuki Shiraki, Chitose Sakai, Yoshiyuki Ishii, and Tomoko Yokota for their technical assistance.

 


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