Dear List,
I am using Voom and the Mean variance trend plot, has a pronounced minimum.
Is this a cause for concern?
Relevant code:
> library(limma)
> library(edgeR)
> library(org.Mm.eg.db)
> targets<-readTargets("targets.txt")
> a1<-read.table("a1.wt.s49_160.mm10.sbrd.counts.txt",header=T,sep="\t",as.is=T,row.names=1)
....
> Counts<-cbind(a1,a2,a3,a4,b1,b2,b3,b4,c1,c2,c3,c4)
> y<-DGEList(counts=Counts, genes=rownames(Counts))
> isexpr<-rowSums(cpm(y)>1) >= 3
>hasannot<-rowSumsis.na(y$genes))==0
> length(hasannot)
> y<-y[isexpr & hasannot,]
> dim(y)
> y<-calcNormFactors(y)
> f<-factor(targets$Target,levels=c("a.wt","b.ldlrko","c.doubko"))
> des<-model.matrix(~0+f)
> colnames(des)<-c("a.wt","b.ldlrko","c.doubko")
> png("liverdia.voomplot.png")
> v<-voomWithQualityWeights(y,design=des,normalization="none",plot=TRUE)
> dev.off()
> sessionInfo()
R version 3.4.2 (2017-09-28)
Platform: x86_64-apple-darwin15.6.0 (64-bit)
Running under: macOS Sierra 10.12.6
Matrix products: default
BLAS: /Library/Frameworks/R.framework/Versions/3.4/Resources/lib/libRblas.0.dylib
LAPACK: /Library/Frameworks/R.framework/Versions/3.4/Resources/lib/libRlapack.dylib
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] parallel stats4 stats graphics grDevices utils datasets methods base
other attached packages:
[1] scatterplot3d_0.3-40 affycoretools_1.48.0 rgl_0.98.1 org.Mm.eg.db_3.4.1 AnnotationDbi_1.38.2 IRanges_2.10.5
[7] S4Vectors_0.14.7 Biobase_2.36.2 BiocGenerics_0.22.1 edgeR_3.18.1 limma_3.32.10
loaded via a namespace (and not attached):
too many to include
>
As far as I know, all of the samples were prepared in the same way. The MDS looks good,
as does hierarchical clustering and pca of the log transformed counts
Thanks and best wishes,
Rich
Richard A. Friedman, PhD
"My Yoga textbook says ’to listen to my body". I am going to listen
to my body, get more sleep, and skip Yoga class” .-Rose Friedman, age 21
