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

We have developed a method to overcome sequencing problems caused by the pr esence of homopolymer stretches, such as polyA/T in cDNA libraries. PolvA t ails are shortened by cleaving before cDNA cloning with type IIS restrictio n enzymes, such as GsuI, placed next to the oligo-dT used to prime the poly A tails of mRNAs. We constructed four rice Cap-Trapper-selected. full-lengt h 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 f or direct sequencing and transcriptional sequencing without the slippage ob served for libraries prepared with currently available methods, thus improv ing sequencing accuracy, operations, and throughput.