[BioC] Read single channel GenePix in limma [was: Analyze miRNA experiment in Bioconductor]

Paul Geeleher paulgeeleher at gmail.com
Wed May 14 15:20:30 CEST 2008 

Hi Sean,

They are Exiqon miRNA Chips.

Thanks,

-Paul

On Wed, May 14, 2008 at 1:29 PM, Sean Davis <sdavis2 at mail.nih.gov> wrote:
> On Wed, May 14, 2008 at 7:54 AM, Paul Geeleher <paulgeeleher at gmail.com> wrote:
>> Hi Gordon,
>>
>> Thanks for you email. I've followed your steps and am getting some output now.
>>
>> One problem though. When should the summarization step occur? What I
>> mean is that, between miRNA and control signals, my GPR file contains
>> about 3000 entries and when I am done with analysis topTable will
>> return all of these individually. But many of the miRNAs have multiple
>> entries in the ".gpr" file. So how, and when, should I go about
>> combining these into one value?
>
> Paul,
>
> What is the manufacturer of these arrays?  The summarization method
> may depend on that somewhat.
>
> Sean
>> On Sun, May 11, 2008 at 4:59 AM, Gordon K Smyth <smyth at wehi.edu.au> wrote:
>>> Dear Paul,
>>>
>>>  The limma User's Guide doesn't discuss how to read single channel data, but
>>> how to do this has been described half a dozen times on this mailing list.
>>> Since limma is designed for two colours, you can fool it by giving two
>>> column names and ignoring the one you don't need.  If you only have the Cy3
>>> channel foreground for example you might use
>>>
>>>   Cy3 <- "F532 Mean"
>>>   RG <- read.maimages(source="genepix",columns=list(R=Cy3,G=Cy3))
>>>
>>>  then
>>>
>>>   RG$R <- NULL
>>>
>>>  to remove the extraneous values.
>>>
>>>  Then RG$G could be given as input to vsnMatrix() and the output analysed
>>> with lmFit().
>>>
>>>  Please don't edit your GenePix files manually, there's no need.  It's prone
>>> to introducing errors and is non-reproducible.
>>>
>>>  The error message "number of items to replace is not a multiple of
>>> replacement length" is not caused by having only one channel.  limma gives a
>>> far more informative message in that case.  The most likely explanation is
>>> that your GenePix files are not of equal lengths.  If that is indeed the
>>> problem, then the limma package doesn't offer any easy solution.  Your only
>>> approach would be to read the files in individually, then align the
>>> expression values yourself.
>>>
>>>  You cannot use read.maimages() with source="imagene" because you do not
>>> have ImaGene files.
>>>
>>>  Best wishes
>>>  Gordon
>>>
>>>
>>>
>>> > Date: Fri, 9 May 2008 15:54:39 +0100
>>> > From: "Paul Geeleher" <paulgeeleher at gmail.com>
>>> > Subject: Re: [BioC] Analyze miRNA experiment in Bioconductor
>>> > To: "Wolfgang Huber" <huber at ebi.ac.uk>
>>> > Cc: bioconductor at stat.math.ethz.ch
>>> >
>>> > Doesn't seem to be anything in the users guide specific to this kind
>>> > of analysis unfortunately.
>>> >
>>> > -Paul
>>> >
>>> > On Thu, May 8, 2008 at 10:31 AM, Wolfgang Huber <huber at ebi.ac.uk> wrote:
>>> >
>>> > > 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
>>> > > > >
>>> > > >
>>> > >
>>> >
>>>
>>
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