Abstract
In alternative expression analysis by sequencing (ALEXA-seq), we developed a method to analyze massively parallel RNA sequence data to catalog transcripts and assess differential and alternative expression of known and predicted mRNA isoforms in cells and tissues. As proof of principle, we used the approach to compare fluorouracil-resistant and -nonresistant human colorectal cancer cell lines. We assessed the sensitivity and specificity of the approach by comparison to exon tiling and splicing microarrays and validated the results with reverse transcription–PCR, quantitative PCR and Sanger sequencing. We observed global disruption of splicing in fluorouracil-resistant cells characterized by expression of new mRNA isoforms resulting from exon skipping, alternative splice site usage and intron retention. Alternative expression annotation databases, source code, a data viewer and other resources to facilitate analysis are available at http://www.alexaplatform.org/alexa_seq/.
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Acknowledgements
We are grateful for funding provided by the University of British Columbia, the Michael Smith Foundation for Health Research, the Natural Sciences and Engineering Research Council, Genome British Columbia, the Terry Fox Foundation, the Canadian Institutes of Health Research, the National Cancer Institute of Canada and the British Columbia Cancer Foundation.
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M.G. and M.A.M. wrote the manuscript. M.G. conducted the analysis, created the figures and participated in all experimental design, laboratory work, programming, and database and website design. O.L.G., R.G., G.R. and R.D.M. contributed to analysis concepts and assisted with programming and code testing. A.S.M., R.G and R.C. assisted with comparisons to other alternative expression tools. M.J.T. provided cell lines and assisted with tissue culture. T.J.P. and Y.-C.H. contributed to laboratory experiments. H.I.L. and A.D. performed the mitochondrial cross species contamination and other quality control analyses. K.T. assisted in the implementation of website hosting and indexing. S.Y.C., J.K.A. and A.A. performed microarray hybridizations and scanning. Y.Z., H.M., T.Z. and M.H. performed Illumina library construction and sequencing. S.C. and J.M. assisted with experimental validations. S.J.M.J. and G.B.M. contributed concepts and experimental designs. I.T.T. provided cell lines and contributed concepts and experimental designs. M.A.M. supported the project and contributed concepts and experimental designs.
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Supplementary Figures 1–11, Supplementary Tables 1–8, Supplementary Results, Supplementary Methods, Supplementary Equations 1–5 (PDF 13812 kb)
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Griffith, M., Griffith, O., Mwenifumbo, J. et al. Alternative expression analysis by RNA sequencing. Nat Methods 7, 843–847 (2010). https://doi.org/10.1038/nmeth.1503
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DOI: https://doi.org/10.1038/nmeth.1503
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