[BioC] Disorderly duplicate spots

James W. MacDonald jmacdon at med.umich.edu
Thu Jan 17 15:27:50 CET 2008

Hi Yannick,

Yannick Wurm wrote:
> Dear List,
> I am a graduate student working with the fire ant Solenopsis invicta.
> We did some two-color cDNA microarrays that I've begun analyzing with  
> limma. But something feels wrong about how I'm doing things: we  
> printed whole clones from a ~25,000 clone cDNA library onto our  
> microarray. Simultaneously, we sequenced our clones. They assemble to  
> ~12,000 transcripts. Many are singlets, but some transcripts are  
> represented by multiple clones (one transcript is represented by 32  
> clones!).
> So during analysis, treating each clone as independent feels wrong.  
> It means:
> 	- correcting for 25,000 multiple tests rather than 10,000, thus  
> reducing my power;
> 	- and not taking into account the technical replication we get by  
> multiple spots on the array.
> The limma manual has a section on Within-Array Replicate Spots. But  
> only mentions what to do for people who have a single duplicate of  
> every spot on their array.
> I'm sure other people have had to deal with this in the past. Do you  
> have any pointers?

This is a not uncommon question on this list, so a search of the list 
archives might be useful. However, to summarize what has been discussed:

1.) limma isn't designed to handle variable numbers of technical replicates.

2.) The alternatives are all suboptimal. You can average the technical 
replicate spots, but this will reduce the variability of the averaged 
measurements and may increase the likelihood that they will appear 
significant. You can ignore the replication (which is what most people 
do with Affy analyses, BTW), but as you noted this will increase your 

3.) Filtering your data using some agnostic criteria such as IQR, 
variance, etc will help with the multiplicity, but there isn't a clean 
way to address variable numbers of technical replicates.



> Thanks & regards,
> Yannick
> --------------------------------------------
>           yannick . wurm @ unil . ch
> Ant Genomics, Ecology & Evolution @ Lausanne
>    http://www.unil.ch/dee/page28685_fr.html
> _______________________________________________
> Bioconductor mailing list
> Bioconductor at stat.math.ethz.ch
> https://stat.ethz.ch/mailman/listinfo/bioconductor
> Search the archives: http://news.gmane.org/gmane.science.biology.informatics.conductor

James W. MacDonald, M.S.
Affymetrix and cDNA Microarray Core
University of Michigan Cancer Center
1500 E. Medical Center Drive
7410 CCGC
Ann Arbor MI 48109

More information about the Bioconductor mailing list