[BioC] About package beadarray

[Wei Shi]

Just adding to Mark's comments: if your data is bead-level data, it is still possible to perform a normexp background correction aided by negative controls. The nec() function in limma performs such a background correction. nec() is the same as neqc() except that it does not include the quantile between-array normalization and log2 transformation steps.

Although neqc() and nec() are designed for pre-processing probe-level data (summarized data), I think using these functions to normalize bead-level data should give the result as good as applying them for the probe-level data, though no such comparisons have been performed. But if you want to work on the bead-level data and also want to use the normexp by control method to normalize them, this might be the way to go.

Cheers,
Wei 

On Jan 10, 2011, at 10:34 PM, Mark Dunning wrote:

> Hi Anand,
> 
> I think I understand the source of your confusion. There are two types
> of correction applied to BeadArray data; one at the bead-level and one
> at the summarized level.
> 
> The bead-level text files created by Illumina's scanning software
> report the intensity of each bead on the array. These have been
> adjusted by subtracting a local background measure calculated using
> nearby pixels on the image. For convenience, the readIllumina function
> in beadarray will read these values, as these are the same values that
> are analysed by Illumina's BeadStudio/GenomeStudio software. We find
> them adequate for most purposes, although a more thorough analysis is
> possible as described in the imageProcessing vignette. If you have
> data exported from BeadStudio they will almost certainly have been
> adjusted in this way.
> 
> However, the backgroundNormalise method (that is now removed) referred
> to in previous emails occurs on a per bead-type basis once data have
> been summarized. It used to be the default normalization method in
> BeadStudio. The quantities subtracted are negative controls
> (bead-types with no target in the genome). As I mention in previous
> posts there are viable alternatives to this method. The neqc method
> you are interested in requires summarized data and (I suspect)
> developed with GenomeStudio data in mind. Therefore it is Ok to use
> data that have been read using readIllumina with this method.
> 
> Hope this helps. Regards,
> 
> Mark
> 
> On Thu, Jan 6, 2011 at 8:37 PM, Anand Patel <acpatel at gmail.com> wrote:
>> Hmm.  This and the image processing file from the current dev version
>> of the package (2.1.3) are seemingly inconsistent?
>> http://bioconductor.org/packages/2.8/bioc/vignettes/beadarray/inst/doc/ImageProcessing.pdf
>> 
>> Suggests that readIllumina always performs the backgroundNormalise process.
>> 
>> http://bioconductor.org/packages/2.8/bioc/vignettes/beadarray/inst/doc/beadlevel.pdf
>> 
>> and what's below suggests that this is not the case anymore.
>> 
>> Could you clarify?  My goal in asking is to take data that's been read with:
>> 
>> and use the neqc normalization process, which requires background
>> correction not have been applied.
>> 
>> Thanks,
>> Anand
>> 
>> On Tue, Dec 14, 2010 at 5:00 AM,  <bioconductor-request at r-project.org> wrote:
>>> Message: 3
>>> Date: Mon, 13 Dec 2010 14:24:45 +0000
>>> From: Mark Dunning <mark.dunning at gmail.com>
>>> To: Changhe Fu <fuchanghe at gmail.com>
>>> Cc: bioconductor at r-project.org
>>> Subject: Re: [BioC] About package beadarray
>>> Message-ID:
>>>        <AANLkTik5PWUwyhTBwWbqsoJcOCW9F68ED5uk8fEYfV=8 at mail.gmail.com>
>>> Content-Type: text/plain; charset=ISO-8859-1
>>> 
>>> Hi,
>>> 
>>> That function used to be in beadarray to implement the background
>>> normalisation that occurs in Illumina's BeadStudio/GenomeStudio.
>>> However, it has been shown in various studies to be inferior to other
>>> methods. See the neqc (limma) or vst (lumi) alternatives available
>>> through the normaliseIllumina function in beadarray.
>>> 
>>> Mark
>>> 
>>> 
>>> On Fri, Dec 10, 2010 at 11:07 PM, Changhe Fu <fuchanghe at gmail.com> wrote:
>>>> Hi, All
>>>> 
>>>> In previous version of package beadarray, I can use the function
>>>> backgroundNormalise(). But in new one, I cann't find it. Is it included in
>>>> some other fuction of normalising?
>>>> 
>>>> Thank you
>>>> 
>>>> --
>>>> Best,
>>>> Changhe Fu
>>>> 
>>>> ? ? ? ?[[alternative HTML version deleted]]
>>>> 
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>> 
> 
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