Question: Draw volcano plot by modifying contrast matrix (lumi package)
0
gravatar for marongiu.luigi
22 months ago by
European Union
marongiu.luigi0 wrote:

Dear all,
I am new to the use of microarray analysis; I have been looking at the use of lumi for the analysis of BeadArray chips.
I have used the tutorial by Du, Feng, Kibbe and Lin (2010) (http://www.bioconductor.org/packages//2.7/bioc/vignettes/lumi/inst/doc/lumi.pdf) to learn the process. In that vignette, the authors use the packages lumi and limma to normalize the data and then fit the linear method (creating an object 'fit') that can identify the most expressed genes. The data come from the example.lumi dataset contained in the package lumiBarnes.
I then used the tutorial by Gillespie (http://bioinformatics.knowledgeblog.org/2011/06/21/volcano-plots-of-microarray-data/) to learn how to draw a volcano plot. It essentially plots the fold change against the -log of the p-value. When I applied this approach to the data of the lumi tutorial, however, I did not obtain the classical 'V' shaped profile but rather a '√' shape. This 'shape alteration' might be inherent to the data, but I reckon it codued be do to the fitting method applied. Both the fitting method and the plotting procedure are based on the topTable function and this is derived from the model.matrix function, thus if the analysis has produced the observed skewness in the data is the model.matrix that I need to modify. In the vignette by Gillespie, the contrast.matrix and contrasts.fit(fit, contrast.matrix) are used instead of model.matrix. 
My questions are then: (a) how can I customize the contrast.matrix? (b) how can I draw a volcano plot of the data? (maybe I simply need to change the fitting method to obtain the expected 'V' shape)

Thank you,

The code follows.

###
# lumi analysis
library(lumi)
library(lumiBarnes)
library(limma)
data("example.lumi")
lumi_t <-lumiT(example.lumi)
lumi_n <- lumiN(lumi_t, method='rsn')
dataMatrix <- exprs(lumi_n)
probeList <- rownames(dataMatrix)
sampleType <- c('100:0', '95:5', '100:0', '95:5') 
design <- model.matrix(~ factor(sampleType))
colnames(design) <- c('100:0', '95:5-100:0')
fit <- lmFit(dataMatrix, design)
fit <- eBayes(fit)
# print most expressed genes
topTable(fit, coef='95:5-100:0', adjust='fdr', number=10)
# volcano plot
gene_list <- topTable(fit, coef='95:5-100:0', number=1000000, sort.by="logFC")
plot(gene_list$logFC, -log10(gene_list$P.Value))
ADD COMMENTlink modified 22 months ago by Gordon Smyth37k • written 22 months ago by marongiu.luigi0
Answer: Draw volcano plot by modifying contrast matrix (lumi package)
0
gravatar for James W. MacDonald
22 months ago by
United States
James W. MacDonald50k wrote:

The results will be identical if you fit the way you have, or if you fit a model that requires a contrasts matrix. Algebraically they are identical. But do note that you (usually) use model.matrix regardless.

design <- model.matrix(~ 0 + factor(sampleType))
contrast <- matrix(c(-1,1), ncol = 1)
fit <- lmFit(dataMatrix, design)
fit2 <- contrasts.fit(fit, contrast)
fit2 <- eBayes(fit2)
volcanoplot(fit2)

 

ADD COMMENTlink written 22 months ago by James W. MacDonald50k
Answer: Draw volcano plot by modifying contrast matrix (lumi package)
0
gravatar for Gordon Smyth
22 months ago by
Gordon Smyth37k
Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia
Gordon Smyth37k wrote:

I think the asymmetric volcano plot is inherent to the data here, not a problem with the processing pipeline. Note that you could have made the volcano plot by

volcanoplot(fit, coef="95:5-100:0")

You might also like to look at:

plotMD(fit, coef="95:5-100:0")

which I think is a lot more informative. It would appear that there a number of genes that are almost absent at 100:0 but strongly expressed at 95:5.

BTW, this data has a clear batch effect (A vs B) that would have to be accounted for in the design matrix, if this was a real analysis.

ADD COMMENTlink modified 22 months ago • written 22 months ago by Gordon Smyth37k
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