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Nucleic Acids Research, 2001, Vol. 29, No. 22 e108
© 2001 Oxford University Press

Establishment of a high throughput EST sequencing system using poly(A) tail-removed cDNA libraries and determination of 36 000 bovine ESTs

Akiko Takasuga, Shinji Hirotsune, Reiko Itoh, Ayako Jitohzono, Harumi Suzuki, Hisashi Aso1 and Yoshikazu Sugimoto*

Shirakawa Institute of Animal Genetics, Odakura, Nishigo, Nishishirakawa, Fukushima 961-8061, Japan and 1Cellular Biology Laboratory, Faculty of Agriculture, Tohoku University, Aoba-ku, Sendai, Miyagi 981-8555, Japan

We determined 36 310 bovine expressed sequence tag (EST) sequences using 10 different cDNA libraries. For massive EST sequencing, we devised a new system with two major features. First, we constructed cDNA libraries in which the poly(A) tails were removed using nested deletion at the 3'-ends. This permitted high quality reading of sequences from the 3'-end of the cDNA, which is otherwise difficult to do. Second, we increased throughput by sequencing directly on templates generated by colony PCR. Using this system, we determined 600 cDNA sequences per day. The read-out length was >450 bases in >90% of the sequences. Furthermore, we established a data management system for analyses, storage and manipulation of the sequence data. Finally, 16 358 non-redundant ESTs were derived from ~6900 independent genes. These data will facilitate construction of a precise comparative map across mammalian species and isolate the functional genes that govern economic traits. This system is applicable to other organisms, including livestock, for which EST data are limited.

* To whom correspondence should be addressed. Tel: +81 248 25 5641; Fax: +81 248 25 5725; Email: kazusugi{at}siag.or.jp+AV588548–AV618892, AV662325–AV668289


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