Pm Intensity Probe Count And Relation To Number Of Affyids Present

Question

[AngryBird]

Hi,

I am working with MoGene-1_0-st-v1 gene microarray in R. This is how my Affydata looks like

AffyBatch object
size of arrays=1050x1050 features (21 kb)
cdf=MoGene-1_0-st-v1 **(34760 affyids)**
number of samples=9
**number of genes=34760**
annotation=mogene10stv1

Now when I do this:

dim(pm(rawData))

the result is

[1] 819041      9

What I cannot understand is the relation between number of affyids and number of PM intensity reads? i.e. 34760:819041

Thanks

Answer 1

[VS]

Each AffyId is for a probeset made up of several PM/MM probes (for ex. In Arabidopsis affy arrays most probesets are made up of 11 probes ). And that is why you see PM probes almost 20 times that of the number of probesets (with their affyIds), so in your mouse array there must be on average around 20-24 probes per probeset.

The following figure might help you better understand the concept (figure taken from http://www.dkfz.de/gpcf/24.html)--

[AngryBird]

I agree, multiple probe sets represent each gene, but what really threw me off, is that the ratio did not come out to be a whole number. That would mean that the probe set count for each affy id is different. Also when I do RMA on these probe sets, they are summarized into a representative value for each gene. Does this mean summarize has an internal logic of calculating a mean for each gene? or does it pick the intensities based on some other logic?

[VS]

Majority of the probesets have same number of probes but not all of them. To find out exactly, you can use following commands in R after loading your .CEL file as an affybatch object under 'affy' package --

ids<-rownames(probes(YourAffyBatch)); ids=strsplit(ids,"_"); ids=paste(sapply(ids,function(x)x[1]),"at",sep="_") ; table(table(ids));

This will give you an output something like the following -- (first row has number of probes in probeset, lower row has the number of probesets carrying that many probes. Ex. There are 19 probesets that contain 8 probes)

8 9 10 11 16 20 48
19 15 16 22725 2 24 3

There are several algorithms to summarize probesets. You can find short descriptions for them in the affy vignette too.

Answer 2

[AngryBird]

This image was taken from http://media.affymetrix.com/support/technical/technotes/gene_1_0_st_technote.pdf is also informative.