[BioC] Analyze miRNA experiment in Bioconductor

[Wolfgang Huber]

Dear Paul,

> Hmm interesting. I might try introducing the extra columns into the
> files and specifying all the values as 0. I can't see why that
> shouldn't work?

It might, but Narendra's suggestion of reading the limma users guide is 
  a worthwhile option to consider.

  Best wishes
	Wolfgang

------------------------------------------------------------------
Wolfgang Huber  EBI/EMBL  Cambridge UK  http://www.ebi.ac.uk/huber

> 
> -Paul
> 
> On Wed, May 7, 2008 at 1:39 PM, Narendra Kaushik
> <kaushiknk at cardiff.ac.uk> wrote:
>> You can specify your red channel like this:
>>
>>  RG <- read.maimages(files,source="genepix",  columns=list(R="F635 Median",G="F532
>>  Median",Rb="B635",Gb="B532"))
>>
>>  I will suggest you read limma guide.
>>
>>  But I think your have data from Imagene package which gives one file for each channel, you can:
>>
>>  files <- targets[,c("FileNameCy3","FileNameCy5")]
>>  RG <- read.maimages(files, source="imagene")
>>
>>  Hope, this helps
>>
>>  Narendra
>>
>>  >>> "Paul Geeleher" <paulgeeleher at gmail.com> 07/05/2008 13:24:01 >>>
>>
>>
>> Hi Deepayan,
>>
>>  Thanks for your reply. I suppose my main concern is how I should read
>>  in the data initially in order to be able to use the normal tools to
>>  analyze the data. Reading the data normally like this:
>>
>>  RG <- read.maimages( files, source="genepix")
>>
>>  Gives the following error:
>>
>>  Error in RG[[a]][, i] <- obj[, columns[[a]]] :
>>   number of items to replace is not a multiple of replacement length
>>
>>
>>  I'm assuming this is down to the fact that the files only contain
>>  intensity data for one color rather than two?
>>
>>  How should I go about reading the data?
>>
>>  Thanks alot,
>>
>>  -Paul.
>>
>>  On Tue, May 6, 2008 at 10:15 PM, Deepayan Sarkar
>>  <deepayan.sarkar at gmail.com> wrote:
>>  > On 5/6/08, Paul Geeleher <paulgeeleher at gmail.com> wrote:
>>  >  > Dear Members,
>>  >  >
>>  >  >  I've inherited a bunch of GenePix files from an miRNA experiment. They
>>  >  >  are single color arrays, ( as opposed to 2 color as is the norm for
>>  >  >  GenePix I think). There is a subset of 7 arrays and I wish to compare
>>  >  >  a group of 4 of these to the other group of 3 and analyze differential
>>  >  >  expression between the two groups. I was hoping somebody could point
>>  >  >  me in the right direction of how I'd go about doing this with
>>  >  >  Bioconductor? Is it possible using the Limma package? Is there any
>>  >  >  code out there to assist me?
>>  >  >
>>  >  >  I've experience in analyzing Affymetrix data using Limma and PUMA, but
>>  >  >  not GenePix, and the Limma Users Guide seems to focus on analyzing two
>>  >  >  dye experiments.
>>  >
>>  >  Any analysis ultimately boils down to some sort of normalization, and
>>  >  the actual differential expression analysis. The second part in limma
>>  >  (lmFit, etc.) can work with any expression matrix, irrespective of
>>  >  whether it's 2-color or 1-color (or affy).
>>  >
>>  >  We have been working with a miRNA array dataset recently, and we used
>>  >  limma to read in the GPR files and do the differential expression
>>  >  analysis (on one channel). For normalization, many of the standard
>>  >  microarray algorithms probably don't make much sense, but VSN seems to
>>  >  work fine.
>>  >
>>  >  We don't really have code (beyond what's already in limma and vsn)
>>  >  that is generally useful; most of the work is in figuring out which
>>  >  rows are of interest (i.e., those representing human miRNAs),
>>  >  combining the replicates (you seem to have four of each), etc. I'm
>>  >  happy to give you more details if you are interested.
>>  >
>>  >  -Deepayan
>>  >