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 Aso, Yoshikazu Sugimoto

    Research output: Contribution to journalArticlepeer-review

    21 Citations (Scopus)

    Abstract

    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.

    Original languageEnglish
    Article numbere108
    JournalNucleic acids research
    Volume29
    Issue number22
    DOIs
    Publication statusPublished - 2020

    ASJC Scopus subject areas

    • Genetics

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