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Question: voom and RPKM for multi-species RNA-seq analysis
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gravatar for Julien Roux
4.5 years ago by
Julien Roux90
Switzerland
Julien Roux90 wrote:
Dear all, I am analyzing RNA-seq data generated from liver samples in two species, human and chimp, with 4 biological replicates in each species. I have mapped the reads to 1-to-1 orthologous exons, and I'm aiming at detecting DE exons between species. After reading the "voom" paper (http://www.statsci.org/smyth/pubs/VoomPreprint.pdf), I am leaning towards using this approach rather than count-based approaches like edgeR or DESeq. One problem that I encounter is that orthologous exons in human and chimp don't always have the same length, so I was looking for some way to correct for exon length when testing for DE. In the voom paper it is mentioned that although voom is based on log-cpm values, it "/can work however just as easily with logged RPKM values in place of log-cpm, because the precision weights are the same for both measures. If the genomic length of each gene is known, then the log-cpm values output by voom can be converted to log-RPKM by subtracting the log-base-2 gene length in kilobases. The downstream analysis is unchanged and will yield identical results in terms of differentially expressed genes and estimated fold changes/." But I think this comment applies to comparisons across genes (e.g., when all samples from an experiment are from the same species), while in my case I wanted to use RPKMs to normalize for differences in gene lengths between samples. Did I miss something? Would you have any advice on how to proceed? Thanks a lot for your help Julien > sessionInfo() R version 3.0.1 (2013-05-16) Platform: x86_64-apple-darwin10.8.0 (64-bit) locale: [1] C attached base packages: [1] parallel stats graphics grDevices utils datasets methods [8] base other attached packages: [1] edgeR_3.2.3 Biobase_2.20.0 BiocGenerics_0.6.0 [4] limma_3.16.5 BiocInstaller_1.10.1 RColorBrewer_1.0-5 loaded via a namespace (and not attached): [1] tools_3.0.1 -- Julien Roux, PhD Gilad lab, Department of Human Genetics, University of Chicago http://giladlab.uchicago.edu/ 920 East 58th Street, CLSC 317, Chicago, IL 60637, USA tel: +1-773-834-1984 fax: +1-773-834-8470 [[alternative HTML version deleted]]
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