Hello Gordon and all,
I am analyzing a RNA-seq experiment, and since I have quite some experience analyzing microarray data using linear models, I decided to go for the 'Voom - Limma' workflow. However, I do have some (practical) queries, and would appreciate feedback on these:
- when transforming the counts data using voom(), is an addition normalization method recommended? E.g. cyclic loess? (normalize.method="cyclicloess"). Note that before applying Voom I already processed the count data with calcNormFactors().
- when applying the eBayes moderated statistical tests, is it still OK to use "trend=TRUE"? fit2 <- eBayes(fit,trend=TRUE). I am asking because voom() also estimates the mean-variance relationship in the data, and I don't want to 'over-correct'. BTW, in the plotSA() graphs I noticed only a minor effect of trend=TRUE.

Thanks,
Guido


library(limma)
samples <- read.delim("samples_descriptions.txt")
counts <- read.delim("count_table_geneLevel_1703.txt", row.names=1)

isexpr <- rowSums(cpm(counts)>1) >= 3
counts <- counts[isexpr,]
#check: > dim(counts)  [1] 10658    18; (62.1% of genes are retained)

d  <- DGEList(counts = counts, group = samples$Condition)
d  <- calcNormFactors(d)

treatment <- as.factor(samples$Condition)
design <- model.matrix(~0+diet)

v <- voom(d,design,plot=TRUE)

# defined contrast matrix, then:
fit <- lmFit(v,design)
fit1 <- contrasts.fit(fit,cont.matrix)

fit2 <- eBayes(fit1,trend=TRUE)
plotSA(fit2)

topTable(fit2)

> sessionInfo()
R Under development (unstable) (2013-11-19 r64265)
Platform: x86_64-w64-mingw32/x64 (64-bit)

locale:
[1] LC_COLLATE=English_United States.1252
[2] LC_CTYPE=English_United States.1252
[3] LC_MONETARY=English_United States.1252
[4] LC_NUMERIC=C
[5] LC_TIME=English_United States.1252

attached base packages:
[1] splines   parallel  stats     graphics  grDevices utils     datasets
[8] methods   base

other attached packages:
[1] edgeR_3.5.28       multtest_2.19.2    Biobase_2.23.6     BiocGenerics_0.9.3
[5] limma_3.19.28

loaded via a namespace (and not attached):
[1] MASS_7.3-29     stats4_3.1.0    survival_2.37-4 tools_3.1.0
>

---------------------------------------------------------
Guido Hooiveld, PhD
Nutrition, Metabolism & Genomics Group
Division of Human Nutrition
Wageningen University
Biotechnion, Bomenweg 2
NL-6703 HD Wageningen
the Netherlands
tel: (+)31 317 485788
fax: (+)31 317 483342
email:      guido.hooiveld@wur.nl
internet:   http://nutrigene.4t.com
http://scholar.google.com/citations?user=qFHaMnoAAAAJ
http://www.researcherid.com/rid/F-4912-2010


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