[limma - v. 17 June 2014] Log2 or not to log2 the data? (Agilent single-channel microarray data)
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FAVI • 0
@favi-7011
Last seen 7.9 years ago
BE

 

Hi all,

I'm working with Agilent single-channel microarray data.

However, it's not clear to me whether raw data should be log2 transformed or not (or if it's intrinsically done "behind the scenes"):

1) In some places (http://matticklab.com/index.php?title=Single_channel_analysis_of_Agilent_microarray_data_with_Limma) the data is not logged at all.

2) Elsewhere (https://stat.ethz.ch/pipermail/bioconductor/2011-August/040543.html) it is said that first quantile normalization is applied to raw intensities and the data has to be (log2)transformed afterwards.

3) On Page 14 (limma's manual) the following paragraphs can be read:

EListRaw. Raw Expression list. A class used to store single-channel raw intensities prior to
normalization. Intensities are unlogged. [...].
EList. Expression list. Contains background corrected and normalized log-intensities.
Usually created from an EListRaw objecting using normalizeBetweenArrays()
.

Does normalizeBetweenArrays() internally (log2)transform the data? [Therefore nothing should be done to the raw data apart from using it as input for normalizeBetweenArrays()]

---------------------------------------------------------------------------------------------------------------------------------------------

So I don't know what am I supposed to do:

a) y <- normalizeBetweenArrays(y,method="quantile")

y$E <- log2(y$E)

b) y$E <- normalizeBetweenArrays(log2(y$E),method="quantile") 
#Transforming the data inside "normalizeBetweenArrays()"

c) Don't change the raw data at all since normalizeBetweenArrays() internally transforms the data (that's my lecture of what's on page 4 of limma's manual (revised 17 June 2014).
 

Any help would be greatly appreciated.


-------------------------------------------------------------------------------------------------------------------------------------------------------------

> sessionInfo()
R version 3.1.1 (2014-07-10)
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] parallel  stats     graphics  grDevices utils     datasets  methods  
[8] base     

other attached packages:
[1] gplots_2.14.2        lattice_0.20-29      vsn_3.34.0          
[4] Biobase_2.26.0       BiocGenerics_0.12.0  limma_3.22.1        
[7] BiocInstaller_1.16.0

loaded via a namespace (and not attached):
 [1] affy_1.44.0           affyio_1.34.0         bitops_1.0-6         
 [4] caTools_1.17.1        gdata_2.13.3          grid_3.1.0           
 [7] gtools_3.4.1          KernSmooth_2.23-12    preprocessCore_1.28.0
[10] zlibbioc_1.12.0      

 

limma logFC normalizeBetweenArrays() • 2.9k views
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Moreover, I noticed that output from "topTable(fit2)" [After having defined fit1 <- lmFit(y, design) / fit2 = contrasts.fit(fit1, contrastMatrix) /
fit2 = eBayes(fit2)] is totally different:

Without log2-transforming: Highest logFC ~ 1.540, lowest logFC ~ -2.586

With log2-transformed data: Highest logFC ~ 0.278, lowest logFC ~ - 0.403
 

(The second approach seems to yield really low values...)

 

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FAVI • 0
@favi-7011
Last seen 7.9 years ago
BE

Problem found.

It seems like, at least in the version I have been using (limma_3.22.1), the function "normalizeBetweenArrays()"  uses raw/background corrected intensities as input and, internally, it automatically (log2)transforms the data (at the same time it performs the normalization (i.e. quantile normalization)).

 

Hope this helps to other people.

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As you've noted, an EListRaw object should contain raw expression values, whereas an EList object should contain logged expression values. If you pass an EListRaw object to normalizeBetweenArrays, the function will detect it as such and log the values before normalization. So, no additional manipulations are necessary. However, note that normalizeBetweenArrays will still return an EList object, i.e., containing normalized logged values.

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