[BioC] limma neqc

[Ina Hoeschele]

Hi Wei,
  thanks again for your response. I am not sure I understand it correctly: I do provide a status vector and I tell neqc that negative control probes are "negative". But I am not sure what regular means. There are positive control probes (biotin, cy3, ERCC,...) and gene or non-control probes which are denoted by "Gene". So do I have to set regular = "Gene"?
Ina
    

----- Original Message -----
From: "Wei Shi" <shi at wehi.EDU.AU>
To: "Ina Hoeschele" <inah at vbi.vt.edu>
Cc: bioconductor at stat.math.ethz.ch
Sent: Wednesday, March 23, 2011 6:14:50 PM
Subject: Re: [BioC] limma neqc

Hi Ina:

	You will have to provide the "status" parameter to neqc then. This is a character vector which tells neqc which probes are regular one, which are negative control and which are positive controls. Regular probes should have a value of "regular" (same with value of the "regular" parameter) and negative controls should have a value of "negative". For positive controls, you can use their original names like "Housekeeping", "Biotin" etc, or you can just use a value of "positive" for all of them. The output of neqc will be a matrix which only includes regular probes.

	Please let me know if you have any further questions.

Cheers,
Wei


On Mar 24, 2011, at 1:15 AM, Ina Hoeschele wrote:

> Hi Wei,
>  thanks much for your quick response. However, I should have mentioned that I am not reading the data using read.ilmn, I am reading and pre-processing the bead-level data via beadarray (doing QC etc.), then summarizing the data and then calling neqc.
> Ina
> 
> ----- Original Message -----
> From: "Wei Shi" <shi at wehi.EDU.AU>
> To: "Ina Hoeschele" <inah at vbi.vt.edu>
> Cc: bioconductor at stat.math.ethz.ch
> Sent: Tuesday, March 22, 2011 6:08:34 PM
> Subject: Re: [BioC] limma neqc
> 
> Dear Ina:
> 
> 	You do not need to specify positive control probes. They were automatically read in when you use read.ilmn function to read in both regular probe data and control probe data. You can use the code below to see the numbers of regular probes, negative control probes and positive control probes:
> 
> x <- read.ilmn(files="probe profile.txt",ctrlfiles="control probe profile.txt", other.columns="Detection") # you will need to changes file names to the names of your files
> table(x$genes$Status)
> 
> 	The neqc function will then perform a quantile normalization for data included in x using all the probes (including regular probes, negative controls and positive controls). The two parameters negctrl and regular tell neqc what the identifiers of regular probes and negative control probes are. The negative control probes play a particular role in the background correction (normexp background correction aided by negative controls). By default, regular probes have an identifier of "regular". Negative control probes by default have an identifier of "negative" which was the identifier used by almost all the BeadChip data we have ever seen. The neqc function should work in most cases with its default setting.
> 
> 	For more details, please refer to the limma user guide for analyzing Illumina BeadChip data (section 11.7).
> 
> 	Hope this helps.
> 
> Cheers,
> Wei
> 
> On Mar 23, 2011, at 6:40 AM, Ina Hoeschele wrote:
> 
>> Hi Gordon et al.,
>>  I am confused about something - I am using limma neqc and according to a recent paper (Shi, Oshlack, Smyth 2010) the quantile normalization should be performed using both positive and negative control probes. But in the arguments for neqc one can specify the identifier for negative control probes and "regular" probes. So I set negctrl="negative" and regular="Gene". But this does not allow me to specify positive control probes?
>> Many thanks, Ina
>> 
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