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Blum, Charles
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@blum-charles-6331
Last seen 9.7 years ago
Hi,
I am running edgeR on 6 RNAseq samples that were generated using the
exact same protocol but are from different Illumina project runs.
In theory, no genes should be differentially expressed. Nevertheless,
edgeR identifies almost 7,000 genes as DE at a FDR rate of 0.1. This
is very puzzling.
I ran edgeR using the classic approach (exactTest) and the glm
approach.
To get an idea of sequencing depth:
Sample:
Project1_sample1 Project1_sample2 Project1_sample3
Project2_sample1 Project2_sample2 Project2_sample3
Total unique annotated read counts: 41,440,190
26,429,859 29,655,944 25,423,167
30,914,059 35,41,714
Could it be due to the variability in sequencing depth between
projects?
Could there anything else in the data or analysis that could violate
any assumptions made by edgeR?
Is there any known problems with the newest version of edgeR?
> sessionInfo()
R version 3.0.2 (2013-09-25)
Platform: x86_64-apple-darwin10.8.0 (64-bit)
locale:
[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8
attached base packages:
[1] splines parallel stats graphics grDevices utils
datasets methods base
other attached packages:
[1] GenomicFeatures_1.14.2 AnnotationDbi_1.24.0
GenomicRanges_1.14.4 XVector_0.2.0
[5] IRanges_1.20.6 biomaRt_2.18.0 edgeR_3.4.2
limma_3.18.7
[9] DESeq_1.14.0 lattice_0.20-24 locfit_1.5-9.1
Biobase_2.22.0
[13] BiocGenerics_0.8.0 gplots_2.12.1 MASS_7.3-29
heatmap.plus_1.3
loaded via a namespace (and not attached):
[1] annotate_1.40.0 Biostrings_2.30.1 bitops_1.0-6
BSgenome_1.30.0 caTools_1.16
[6] DBI_0.2-7 gdata_2.13.2 genefilter_1.44.0
geneplotter_1.40.0 grid_3.0.2
[11] gtools_3.1.1 KernSmooth_2.23-10 RColorBrewer_1.0-5
RCurl_1.95-4.1 Rsamtools_1.14.2
[16] RSQLite_0.11.4 rtracklayer_1.22.0 stats4_3.0.2
survival_2.37-4 tools_3.0.2
[21] XML_3.95-0.2 xtable_1.7-1 zlibbioc_1.8.0
> packageDescription('edgeR')$Maintainer
[1] "Mark Robinson <mark.robinson at="" imls.uzh.ch="">, Davis
McCarthy\n<dmccarthy at="" wehi.edu.au="">, Yunshun Chen <yuchen at="" wehi.edu.au="">,\nGordon Smyth <smyth at="" wehi.edu.au="">"
Thanks,
Charles
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