Weird MAplot with DEGs lying along the diagonals
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@federico-alessandro-25010
Last seen 6 months ago

Dear Bioconductor users,

I'm currently using limma to reprocess some (not so) old microarray data originally acquired using the following platform: Agilent-026652 Whole Human Genome Microarray 4x44K v2 comparing a tumor sample and the matched "healthy" region nearby, in the same biopsy.

I'm interested in differential gene expression, as usual, but I get really weird MAplots (and quite compressed BoxPlots) in the preprocessing step. Here is an example: BoxPlots of Raw data MAplot of Raw data for one tumor sample vs its matched healthy sample BoxPlots of Background-subtracted data MAplot of Background-subtracted data for one tumor sample vs its matched healthy sample BoxPlots of Quantile-Normalized data MAplot of Quantile-Normalized data for one tumor sample vs its matched healthy sample

It seems like the typical bulk of the highly-expressed-unchanged genes is completely missing, while all the DEGs have been "pushed" towards the two diagonals of the plot... Never seen something like this before... Do you think this is biologically reliable? Maybe some problem with hybridization or scanning?

Any help/suggestion/comment from anyone more experienced than me is welcome!

Thank you so much!

I think the problem is in the biology or in the wet-lab step, and not in the coding. However, here is the (very essential and almost standard) code I used:

library(limma)
targets = readTargets("Targets.txt", row.names = "SampleNumber")
raw = read.maimages(targets, source = 'agilent.median', green.only = TRUE)
   # Here I generate some QC plots of Raw data
raw_BGcorrected = backgroundCorrect(raw, method = "normexp", offset = 50)
   # Here I generate some QC plots of BG-subtracted data
raw_BGandNormalized = normalizeBetweenArrays(raw_BGcorrected, method = "quantile")
   # Here I generate some QC plots of Normalized data
limma Microarray AgilentChip MA-plot Normalization • 296 views
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@gordon-smyth
Last seen 4 hours ago
WEHI, Melbourne, Australia

Looks to me like the array hybridizations have failed. The arrays seem to have essentially no signal, with all probe intensities at the same low level except for a few outliers on each array.

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Thank you for the comment. An hybridization failure was also my suspect, but I feel better with the expert's opinion :-) By the way, the analysis of the Agilent control probes within the array seems to confirm that. Thank you again! bye

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