Removal of PolyA Tails from Full-Length cDNA Libraries for High-Efficiency Sequencing | Zendy

Yûkô Shibata | Zendy; Piero Carninci | Zendy; K Sato | Zendy; Norihito Hayatsu | Zendy; Toshiyuki Shiraki | Zendy; Yoshiyuki Ishii | Zendy; Takahiro Arakawa | Zendy; Ayako Hara | Zendy; N. Ohsato | Zendy; Masaki Izawa | Zendy; Katsunori Aizawa | Zendy; Masayoshi Itoh | Zendy; Kazuhiro Shibata | Zendy; Akira Shinagawa | Zendy; Jun Kawai | Zendy; Yoshimi Ota | Zendy; Shoshi Kikuchi | Zendy; Naoki Kishimoto | Zendy; Masami Muramatsu | Zendy; Yoshihide Hayashizaki | Zendy

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Removal of PolyA Tails from Full-Length cDNA Libraries for High-Efficiency Sequencing

Author(s) -

Yûkô Shibata,

Piero Carninci,

K Sato,

Norihito Hayatsu,

Toshiyuki Shiraki,

Yoshiyuki Ishii,

Takahiro Arakawa,

Ayako Hara,

N. Ohsato,

Masaki Izawa,

Katsunori Aizawa,

Masayoshi Itoh,

Kazuhiro Shibata,

Akira Shinagawa,

Jun Kawai,

Yoshimi Ota,

Shoshi Kikuchi,

Naoki Kishimoto,

Masami Muramatsu,

Yoshihide Hayashizaki

Publication year - 2001

Publication title -

biotechniques

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 0.617

H-Index - 131

eISSN - 1940-9818

pISSN - 0736-6205

DOI - 10.2144/01315st04

Subject(s) - complementary dna , cdna library , cloning (programming) , biology , genomic library , dna sequencing , computational biology , contig , restriction enzyme , genetics , microbiology and biotechnology , library , deep sequencing , gene , base sequence , computer science , genome , 16s ribosomal rna , programming language

We have developed a method to overcome sequencing problems caused by the presence of homopolymer stretches, such as polyA/T, in cDNA libraries. PolyA tails are shortened by cleaving before cDNA cloning with type IIS restriction enzymes, such as GsuI, placed next to the oligo-dT used to prime the polyA tails of mRNAs. We constructed four rice Cap-Trapper-selected, full-length normalized cDNA libraries, of which the average residual polyA tail was 4 bases or shorter in most of the clones analyzed. Because of the removal of homopolymeric stretches, libraries prepared with this method can be used for direct sequencing and transcriptional sequencing without the slippage observed for libraries prepared with currently available methods, thus improving sequencing accuracy, operations, and throughput.

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