[BioC] Coercing matrix into expression set, for normalization of only subsets of miRNAs (affy miRNA3.0)

James W. MacDonald jmacdon at uw.edu
Fri Oct 25 23:41:41 CEST 2013

Hi Dana,

On Friday, October 25, 2013 5:14:36 PM, Dana Most wrote:
> Hi Jim,
> Thank you for your response.
> I am trying to separate the mature and premature microRNAs because
> they have different hybridization characteristics. I thought it would
> be more 'statistically correct' to normalize them separately since
> these two populations are different. If I don't intend to compare the
> premature and mature microRNAs to each other, are there any
> disadvantages of separating them? except for the obvious reason - that
> there is no function available for easy use and I am hardly a programmer.

What do you mean by 'different hybridization characteristics'? 
Hybridization to the array, or something biological?

If you mean biologically different, that is not relevant to the 
normalization. The only thing that is relevant to the normalization is 
that the relative amount of transcript doesn't change for most miRNAs, 
and that the distribution of the data is similar between arrays. Any 
biological consideration is moot. If it weren't, the quantile 
normalization wouldn't be reasonable for mRNA arrays either.

If you mean that the hybridization to the array is different, then in 
what way? All the probes are just 25-mers. I guess you could argue that 
the hairpin transcripts, being longer will hybridize differently, but 
that still doesn't matter. The only assumptions being made are 
distributional, so if one transcript tends to bind better or worse than 
another, it doesn't matter. Again, if it did, quantile normalization 
wouldn't be useful for mRNA arrays either.

> So there's no easy way to coerce a data matrix (or a subset of the
> data) into an ExpressionFeatureSet structure to satisfy the rma function?
> Maybe I can convert the matrix into an ExpressionSet using Biobase, do
> you know if there's a way to to convert the ExpressionSet into an
> ExpressionFeatureSet?

No, you can't go back. What you have to understand is that each miRNA 
has a certain number of probes that measure the expression of that 
miRNA. When you run rma(), you are summarizing the expression of all 
the probes that measure a given miRNA in order to come up with a single 
value for each miRNA. An ExpressionSet isn't designed to handle the 
data in an ExpressionFeatureSet. In addition, the summarization 
requires that you know _which_ of the probes belong to a given probeset 
(where the probeset gives an idea of the expression for a single 
miRNA). If you mess with the data in an ExpressionFeatureSet, you also 
have to change the pd.info package to reflect those changes.

Long story short, I think you are worrying about something that is not 
worth worrying about. You can certainly try to do something to 
normalize these probes differently but in the end I doubt it will make 
a difference, and almost surely won't have been worth the time spent.



> Thank you so much for your help!
> Dana
> On Thu, Oct 24, 2013 at 9:07 AM, James W. MacDonald <jmacdon at uw.edu
> <mailto:jmacdon at uw.edu>> wrote:
>     Hi Dana,
>     On Wednesday, October 23, 2013 12:04:31 AM, Dana Most wrote:
>         Dear All,
>         How can I transform/coerce a gene expression matrix into an
>         expression set?
>         I'm using affy miRNA 3.0 data and I would like to normalize
>         only a subset of
>         the samples (i have 4 groups of samples and would like to
>         choose 2) and only a
>         subset of microRNAs (I have mature and premature microRNAs and
>         they should not
>         be normalized together).
>         It should look something like this:
>         affyExpressionFS <- read.celfiles(celFiles,
>         pkgname="pd.mirna.3.0")
>         data = exprs(affyExpressionFS)
>         data = data[1:1000,1:20]
>     Why do you think the first 1000 rows are useful here? Is this just
>     supposed to be an example?
>         exprsData = coerce data into expression set
>         rma(exprsData)
>     You can't run rma on an ExpressionSet, as an ExpressionSet is
>     intended to contain summarized data. Instead you need to use an
>     ExpressionFeatureSet object (which is what you are getting your
>     matrix of data out of).
>     That said, you will have to do some serious coding if you want to
>     accomplish this. Right now there is no easy way (that I know of -
>     Benilton might correct me here) to subset to a particular set of
>     probes. You can check out the oligo source from subversion and
>     make whatever changes you want. There is even a 'subset' argument
>     that is for future use that you could implement if you want.
>     But this leads me to your original rationale for wanting to do
>     this, where you state that mature and precursor miRNAs should not
>     be normalized together. I am not sure why you would think this,
>     and I am pretty sure you are wrong.
>     You could argue that the hairpin miRNAs are fundamentally
>     different from the mature miRNAs (which I suppose they are), but
>     that has nothing to do with normalization. For the normalization
>     to be reasonable, you have to fulfill two criteria. First, most
>     probes should not be differentially expressed between samples, and
>     second, the underlying distributions of the data should not be
>     completely dissimilar.
>     This has nothing to do with what the probes are supposed to
>     measure, nor whether or not the probes are even measuring anything
>     at all. So I don't see any real reason to separate the hairpin
>     from mature miRNAs prior to normalizing.
>     Best,
>     Jim
>         Also, I would like to use array quality metrics package on
>         exprsData
>         arrayQualityMetrics(__expressionset = exprsData,
>         outdir = "exprsData",force = FALSE, do.logtransform = FALSE)
>         Thank you,
>         Dana
>         _________________________________________________
>         Bioconductor mailing list
>         Bioconductor at r-project.org <mailto:Bioconductor at r-project.org>
>         https://stat.ethz.ch/mailman/__listinfo/bioconductor
>         <https://stat.ethz.ch/mailman/listinfo/bioconductor>
>         Search the archives:
>         http://news.gmane.org/gmane.__science.biology.informatics.__conductor
>         <http://news.gmane.org/gmane.science.biology.informatics.conductor>
>     --
>     James W. MacDonald, M.S.
>     Biostatistician
>     University of Washington
>     Environmental and Occupational Health Sciences
>     4225 Roosevelt Way NE, # 100
>     Seattle WA 98105-6099

James W. MacDonald, M.S.
University of Washington
Environmental and Occupational Health Sciences
4225 Roosevelt Way NE, # 100
Seattle WA 98105-6099

More information about the Bioconductor mailing list