Abstract
We report an alternative approach to transcriptome sequencing for the Illumina Genome Analyzer, in which the reverse transcription reaction takes place on the flowcell. No amplification is performed during the library preparation, so PCR biases and duplicates are avoided, and because the template is poly(A)+ RNA rather than cDNA, the resulting sequences are necessarily strand-specific. The method is compatible with paired- or single-end sequencing.
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Acknowledgements
We thank N. Bason and M. Quail for preparing the standard RNA-seq library, J. Burton for coordinating the sequencing of standard RNA-seq libraries, and J. Ule and M. Wollerton for advice on RNA ligation. This work was supported by the Wellcome Trust grant (WT079643).
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D.J.T. and T.W.B.O. devised the project; L.M. and D.J.T. devised the experimental protocols; L.M. and E.M.S. planned and carried out laboratory work; R.M.A. and K.D.J. performed data analysis; P.D.E. performed microarray work; and C.F.L. oversaw analysis and array work. D.J.T., J.E.C. and L.M. wrote the manuscript.
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T.W.B.O. is an Illumina employee, shareholder and share option holder.
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Mamanova, L., Andrews, R., James, K. et al. FRT-seq: amplification-free, strand-specific transcriptome sequencing. Nat Methods 7, 130–132 (2010). https://doi.org/10.1038/nmeth.1417
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DOI: https://doi.org/10.1038/nmeth.1417
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