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Removal of polyA tails from full-length cDNA libraries for high-efficiency sequencing

Authors :
Y. Shibata
N. Hayatsu
N. Ohsato
Takahiro Arakawa
Yoshihide Hayashizaki
Piero Carninci
Masami Muramatsu
Kazuhiro Shibata
Yoshiyuki Ishii
Katsunori Aizawa
S. Kikuchi
Akira Shinagawa
Naoki Kishimoto
A. Hara
Jun Kawai
Masaki Izawa
Kenjiro Sato
Masayoshi Itoh
Toshiyuki Shiraki
Y. Ota
Source :
ResearcherID, Scopus-Elsevier
Publication Year :
2001

Abstract

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.

Details

ISSN :
07366205
Volume :
31
Issue :
5
Database :
OpenAIRE
Journal :
BioTechniques
Accession number :
edsair.doi.dedup.....fd5809f1e0767f2e264ded12e6adffba